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LEGUME

The journal of the International Legume Society

Issue 5 • October 2014

Legumes of Southeast EuropeThe hitchhikers along an ancient Eurasian crop highway

PERSPECTIVES

ISSN2340-1559 (electronic issue)

Quarterly publicationJanuary, April, July and October

(additional issues possible)

Published byInternational Legume Society (ILS)

Co-published byInstitute of Field and Vegetable Crops, Novi Sad, Serbia

CSIC, Institute for Sustainable Agriculture, Córdoba, Spain

Publishing DirectorDiego Rubiales

CSIC, Institute for Sustainable Agriculture, Córdoba, Spain [email protected]

Office and subscriptionsCSIC, Institute for Sustainable Agriculture

International Legume SocietyApdo. 4084, 14080 Córdoba, Spain

Phone: +34957499215 • Fax: +34957499252 [email protected]

Front cover art:The SEELEGUMES project logo by Aleksandar Mikić

IMPRESSUM

Editorial Board

Editor-in-ChiefDiego Rubiales

Assistant EditorsMike Ambrose (genetic resources), Paolo Annicchiarico(lucerne), Birte Boelt (seed production), Beat Boller(clovers), Ousmane Boukar (cowpea), Judith Burstin (pea), MarinaCarbonaro (pharmaceutical uses), Branko Ćupina (non-fooduses), Vuk Đorđević (soybean), Gérard Duc (faba bean), Noel Ellis(genomics), Sara Fondevilla (bioinformatics), Bernadette Julier(breeding), Branislav Kovačević (black locust), Kevin McPhee(genetics), Aleksandar Medović (archaeobotany), Aleksandar Mikić(vetches), Teresa Millán (chickpea), Fred Muehlbauer(lentil), Ramakrishnan Nair (food uses), Pádraig O’Kiely (feeduses), Dejan Pajić (scientometrics), Diego Rubiales (bioticstress), Christophe Salon (phenomics), Marta Santalla (commonbean), Petr Smýkal (wild relatives), Frederick L. Stoddard (abioticstress), Wojciech Święncicki (lupins), Richard Thompson (Medicagotruncatula), Rajeev Varshney (pigeon pea), Carlota Vaz Patto(vetchlings), Tom Warkentin (quality), Christine Watson(agronomy), Ping Wan (adzuki bean), Daniel Wipf (symbiosis)

Technical EditorAleksandar Mikić

Institute of Field and Vegetable Crops, Novi Sad, [email protected]

FOR AUTHORS

Legume Perspectives is an international peer-reviewed journal aimingto interest and inform a worldwide multidisciplinary readership onvery different aspects of the research and use of all kinds of legumeplants and crops. Its scope ranges from biodiversity, genetics andbreeding to agronomy, animal production, human nutrition andhealth and economy.

The issues of Legume Perspectives are usually thematic and devoted tospecific crop or topic. They are edited by guest editor, chosen by theEditor-in-Chief and Assistant Editors, who solicit the articles, mostlyreviews, from selected authors. The original research articles are alsowelcome, but are considered for publication by the Editorial Board.

Regardless of the article category, Legume Perspectives prefers aclear, simple and comprehensive writing style that would make itsarticles interesting and useful for both academic and amateuraudience. Your article is expected to assist in the exchange ofinformation among the experts in various fields of legume research.

Submit either longer (900-1,100 words + up to 3 tables, figures orphotos + up to 10 references) or shorter (400-500 words + 1table, figure or photo + up to 4 references) manuscripts. TheEditorial Board of Legume Perspectives may allow any variation inlength or structure from case to case.

The manuscripts for Legume Perspectives should be prepared inMicrosoft Office Word, using Times New Roman font, 12 pointssize and single spacing. Please provide each manuscript with a 100-word abstract and 4-6 key words. The references should follow thestyle of any published paper in this issue, be given in full and listedalphabetically. The tables may be incorporated in themanuscript, while figures, photographs or drawings should besubmitted separately as jpg files with a resolution of at least 600 dpi.

Please send your prepared manuscripts for publishing in LegumePerspectives, as well as any ideas or questions, to Technical Editor([email protected]).

Interested in grain, forage and other legume research?Join Legume Society today!

[email protected]

3Legume Perspectives Issue 5 • October 2014

s

elcome to theIssue 5 of Legume

Perspectives! Unlike the previous four that were devoted to individual legume crops, this one brings you the articles resulting mostly from one project. Its name is Sustainable preservation of indigenous South East European legumes and their traditional food and feed products (SEELEGUMES) and it was carried out during 2011 and 2012 within the SEE-ERA programme of the European Union and under the auspices of its Seventh Framework Programme (FP7). The articles in this issue are authored by majority of the project participants and deal with diverse legume plants and crops, as well as with various research topics. We deeply believe that this issue of Legume Perspectives, deliberately illustrated to a perhaps greater extent than usual, will bring you an impression of the genuine beauty and richness of the legume flora of Southeastern Europe. Enjoy your reading!

Branko Ćupina andAleksandar Mikić

Managing Editors ofLegume Perspectives Issue 5

WWCONTENTSEDITORIAL

CARTE BLANCHE

4 Branko Ćupina, Aleksandar Mikić: Where Europe meets Asia and the Present mingles with the Past

RESEARCH

5 Aleksandar Medović, Aleksandar Mikić: Archaeobotanical findings of annual and other legumes in Serbia

7 Roman Hovsepyan: Pulses cultivated in prehistoric periods at the territory of Armenia: Short review of present archaeobotanical data

9 Petr Smýkal, Aleksandar Medović, Živko Jovanović, Nemanja Stanisavljević,Bojan Zlatković, Branko Ćupina, Vuk Đorđević, Aleksandar Mikić: Molecular analysis of ancient DNA isolated from charred pea (Pisum sativum) seeds found at an Early Iron Age settlement in southeast Serbia

11 Bojan Zlatković, Stefan Bogosavljević, Aleksandar Mikić, Branko Ćupina: Legumesdiversity in the gorges of Carpathian-Balkan Mountain arc in Serbia

14 Aleksandar Simić, Savo Vučković: Pasture and meadow legumes in Serbia

16 Sokrat Jani, Agim Canko, Milto Hyso: Sustainable preservation of indigenous Albanian legumes and their traditional use

19 Aleksandar Mikić, Branko Ćupina, Bojan Zlatković, Petr Smýkal, Svetlana Antanasović, Vuk Đorđević, Aleksandar Medović, Đorđe Krstić, Vojislav Mihailović: Collecting, characterisation and evaluation of ‘tall’ pea (Pisum sativum subsp. elatius) in southeastern Serbia

23 Branko Đurić: Reintroduction of faba bean (Vicia faba L.) in the Republic of Srpska

24 Siyka Angelova, Mariya Sabeva, Yana Guteva: Local plant genetic resources of fababean (Vicia faba) in Bulgaria

26 Cengiz Toker, Jens Berger, Abdullah Kahraman, Abdulkadir Aydogan, Canan Can, Bekir Bukun, R. Varma Penmetsa, Eric J. Von Wettberg, Douglas R. Cook: Cicer reticulatumLadizinsky, progenitor of the cultivated chickpea (C. arietinum L.)

28 Vladimir Meglič, Jelka Šuštar-Vozlič, Marko Maras: South East European (SEE) autochtonous Phaseolus bean germplasm genetic diversity and reintroduction of traditional landrace

30 Lana Zorić, Jadranka Luković, Dunja Karanović, Aleksandar Mikić: Lignified tissues in vegetative organs of wild-growing Trifolium L. forage species

32 Margarita Vishnyakova, Alla Solovyova, Andrey Sabitov, Pavel Chebukin, Marina Burlyaeva: Economic value of perennial vetchlings (Lathyrus L.) species from the Russian Far East

34 Janna Akopian, Andrey A. Sinjushin, Ivan Gabrielyan, Gayane G. Shaboyan: On some biomorphological peculiarities of seedlings of Vavilovia formosa (Stev.) Fed. (Fabaceae)

36 Sergio J. Ochatt, Catherine Conreux: In vitro biotechnology approaches now available for ‘beautiful’ vavilovia (Vavilovia formosa)

37 Tihomir Čupić, Marijana Tucak, Aleksandar Mikić: Nutritive value of grass pea (Lathyrussativus)

39 Nemanja Stanisavljević, Živko Jovanović, Tihomir Čupić, Jovanka Lukić,Jovanka Miljuš Đukić, Svetlana Radović, Aleksandar Mikić: The effect of cooking and enzymatic digestion on phenolic content of grass pea seeds flour extracts

41 Đorđe Malenčić, Biljana Kiprovski, Branko Ćupina, Aleksandar Mikić: Screening for dietary phenolics and antioxidant capacity of faba bean (Vicia faba) and vetches (Vicia spp.)

PROJECTS

42 Branko Ćupina, Aleksandar Mikić: SEELEGUMES: One genuine piece in the jigsaw puzzle of the European legume biodiversity

EVENTS

43 Second International Legume Society Conference, Tróia, Portugal, 12-14 October 2016

IN MEMORIAM

47 Professor Reid G. Palmer


4

Where Europe meets Asia and the Present mingles with the Past

bove East and West” is an old religious, phylosophicand poetic term denoting what in a more politicallanguage, is often denoted as southeastern Europeor, from a geographic point of view, the Balkan

Peninsula. This region was truly, as designated in the subtitle of thisissue of Legume Perspectives, one of the main routes Neolithic cameto Europe, some ten thousand years ago, when Europe had broke freefrom the cold dungeon of the last Ice Age.

Indeed, the first farmers, coming from the parts of thefamous Fertile Crescent in Near East via Asia Minor, crossed over intoour home continent in present Thrace and quickly sped forward up theflow of the river Danube into other regions. They bore with themselvestheir revolutionary culture of tilling soil and cultivating plants.According to the present knowledge on these earliest days of crops inEurope, along with three cereals, namely einkorn, emmer andbarley, and flax, there were four pulses: chickpea, lentil, pea and bittervetch. In parallel, the wild flora, that once had been present and thathad perished under the glaciers and their lethal breath, began to returnto the places it had inhabited eons ago, enriching the landscapes ofSoutheastern Europe once more and making them both beautiful andgenuine. In the end, the crops from other continents, such as Phaseolusbeans, found their new home here and became indispensable in humandiets. All this biodiversity, both agricultural and wild, have alwayscomprised a considerable number of legumes and endured the passingmillennia.

Today, it is none else but the Man himself who is themain threat to the survival and diversity of all those countless legumecrops and their wild relatives. Travelling along this ancient Eurasianhighway, one meets the legume hitchhikers less often and theirmilestones have become rather rare. An uncontrolled development ofindustry and urbanisation pay their toll every day by making furtherlife of one legume taxon nearly every day. Our task, asagronomists, geneticists, breeders, botanists, biologists orarchaeobotanists, is to work persistently and as hard as possible towardsone common goal: to preserve and conserve a treasury we have beenblessed with and multiply its benefits for the health and the wealth ofour own children. ■

_________________________________________________________________________________________________________

1University of Novi Sad, Faculty of Agriculture, Department of Field and Vegetable Crops, Novi Sad, Serbia ([email protected])2Institute of Field and Vegetable Crops, Novi Sad, Serbia ([email protected])

Carte blanche to…

...Branko Cupina1

andAleksandar

Mikic2

“A

Legume Perspectives Issue 1 • January 20134Legume Perspectives Issue 5 • October 2014

,

,

10

Archaeobotanical findings of annual and other legumes in Serbiaby Aleksandar MEDOVIĆ1* and Aleksandar MIKIĆ2

Abstract: The oldest archaeological findingsof chickpea (Cicer arietinum), lentil (Lensculinaris), pea (Pisum sativum) and bitter vetch(Vicia ervilia) date back as early as from 9thmillennium BC and are mostly in Syria andIsrael. Together with few cereals and othercrops, grain legumes entered Europe via AsiaMinor. Serbia is rather rich inarchaeobotanical remains of cultivated andwild legumes. Lentil and pea may beregarded as the most important in this regiontransgressing from the Balkan to CentralEurope, being present in most of theexcavated material and with Gomolava asone of the most ancient, dated back to 6thmillennium BC. The most interesting find ofgrain legume crops in Serbia comes from thefortified hill of Hissar, near presentLeskovac, dated to 11th century BC, withseveral thousands of the charred seeds ofboth bitter vetch and pea.Key words: archaeobotany, charredseeds, crop history, pulse crops, Serbia

Chickpea (Cicer arietinum L.), lentil (Lensculinaris Medik.), pea (Pisum sativum L.) andbitter vetch (Vicia ervilia (L.) Willd.) areamong the first domesticated plant species inthe world. The oldest archaeological findingsfrom 9th millennium BC are mostly in Syriaand Israel (3). From Near East, agriculturebegan to spread in all directions. Togetherwith few cereals and other crops, grainlegumes entered Europe via Asia Minor.

_________________________________________________________________________________________________________

1Museum of Vojvodina, Novi Sad, Serbia ([email protected])2Institute of Field and Vegetable Crops, Novi Sad, Serbia

Serbia is rather rich in archaeobotanicalremains of cultivated and wild legumes, withthe territory of its northern Province ofVojvodina as rather well-studied (Fig. 1). Asmay be seen, chickpea was mostly absent atthe majority of the studied archaeologicalsites. This may be explained by a probableloss of winter-hardy genotypes during thechickpea distribution to more northernlatitudes, although this crop, as the othercool season legumes, has a considerabletolerance to the intensity and length of lowtemperatures. All this most likely resulted ina much more uncertain cultivation thanthose of pea or lentil (Fig. 2).

On the other hand, lentil and pea may beregarded as the most important in this regiontransgressing from the Balkan to CentralEurope, being present in most of theexcavated material and with Gomolava asone of the most ancient, dated back to 6thmillennium BC. Faba bean (Vicia faba L.) andcommon vetch (Vicia sativa L.) appearedduring the Early Iron Age, grass pea(Lathyrus sativus L.) during La Tène andfenugreek (Trigonella phoenum-graecum L.) inthe Roman period. As for the weedy legumeflora, the oldest remains belong to the generaCoronilla L. and Vicia L.

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Figure 1. Soil map of the Serbian Province of Vojvodina, with the most important archaeobotanical finds of pulse crops: alluvial deposit (light green), typical loess (brown),terrace loess (yellow), aeolian sand (red), gravel, sand and clay (pink) and mountains (dark green)

STARČEVO

OPOVO

GOMOLAVAHRTKOVCI - VRANJ

ŽIDOVAR

FUEDVAR

GRADINA NA BOSUTU

ČURUG –STARI VINOGRADI

ČARNOK –BAČKO DOBRO POLJE

9

The most interesting find of grain legumecrops in Serbia, namely pea and bittervetch, comes from the fortified hill ofHissar, near present Leskovac, dated to 11thcentury BC (1). Unlike the vast majority ofarchaeological sites, where cereals are muchmore dominant, here several thousands ofthe charred seeds of both bitter vetch andpea were identified (2) (Fig. 3). The reasonwhy the Hissar population was so fond ofpulses in their diets remainsunanswered, although this tradition of theexcellence in preparing pulse-based meals inthe region have survived until today.

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Legume Perspectives Issue 1 • January 2013

References(1) MedovićA, JovanovićŽ, Stanisavljevic N, RadovićS, MikićA, ĐorđevićV, Ćupina B (2010) An archaeobotanical and molecular fairy tale about the early Iron Age Balkan princess and the charred pea. Pisum Genet 42:35-38(2) MedovićA, Mikić A, Ćupina B, JovanovićŽ, Radović S et al (2011) Pisum & ervilia Tetovac – Made in Early Iron Age Leskovac. Part One. Two charred pulse crop storages of the fortified hill fort settlement Hissar in Leskovac, South Serbia. Ratar Povrt 48:219-226(3) Tanno K, Willcox G (2006) The origins of cultivation of Cicer arietinum L. and Vicia faba L.: Early finds from Tell el-Kerkh, north-west Syria, late 10th millennium B.P. Veg Hist Archaeobot 15:197-204


Figure 2. Presence of the ancient Eurasian pulse crops in the archaeological sites in Vojvodina, Serbia

Site Period

Cic

erar

ietin

um

Lath

yrus

sativ

us

Lens

cul

inar

is

Pisu

msa

tivum

Vic

iaer

vilia

Vic

iafa

ba

Vic

iasa

tiva

Trig

onel

lafo

enum

-

Cor

onill

ava

ria

Cor

onill

a-Ty

pe

Lath

yrus

niss

olia

Trig

onel

la

Trifo

lium

prat

ense

Trifo

lium

-Typ

e

Vic

ia-T

ype

Vic

ia/L

athy

rus-

Čarnok - BačkoDobro Polje

La Tène

Čurug -Stari vinogradi

Roman period (Barbaric)

FeudvarBronze & Early Iron Age

Gomolava

Neolithic

Eneolithic

Early Iron Age

La Tène

Gradina naBosutu

Early Iron Age

Hrtkovci - Vranj Roman period

Kalakača Early Iron Age

Opovo Neolithic

Starčevo Neolithic

Židovar

Bronze Age

La Tène

Figure 3. Charred seeds of pea (left) and bitter vetch (right) from Hissar, souteastearnSerbia, 11th century BC

10

Pulses cultivated in prehistoric periods at the territory of Armenia: Short review of present archaeobotanicaldataby Roman HOVSEPYAN

Abstract: The most common cultivatedpulses in the territory of Republic ofArmenia (South Caucasus) in prehistorictimes were lentil (Lens culinaris) and pea(Pisum sativum). These crops are recordedalready in early agricultural period, namely inthe Late Neolithic - Early/MiddleChalcolithic (end of 7th millennium -beginning of 5th millennium cal BC)settlements in the Ararat valley. There arethree main types of archaeobotanicalfindings of pulses: charred seeds from softarchaeological sediments (soil), theimpressions of pods valves and sometimesseeds in the clay-made walls of dwellings andstructures. Charred seeds fromarchaeological sediments are generallyinterpreted as remains of food, andimpressions of pods valves are remains ofcrop threshing waste used as temperingmaterial in building clay. Findings of pulses(pea, lentil, grass pea) from Late Chalcolithicperiod (43rd-33rd centuries BC) arerepresented with charred seeds and they aremuch less than cereals remains.Key words:Armenia, archaeobotany, charred seeds, crophistory, pulse crops

_________________________________________________________________________________________________________

Institute of Archaeology and Ethnography NAS, Yerevan, Armenia ([email protected])

Lentil (Lens culinaris Medik.) and pea (Pisumsativum L.) were the most common cultivatedpulses in the territory of Republic ofArmenia (South Caucasus) in prehistorictimes. In this territory pulses are recordedalready in early agricultural, Late Neolithic –Early/Middle Chalcolithic period (end of 7thmillennium - beginning of 5th millennium calBC) settlements in the Ararat valley. In theearliest stage of agriculture in the territory ofArmenia, pulses, such as small-seededlentil, bitter vetch and possibly pea (Figure1), had important role in food economy andagrarian culture of local population: remainsof pulses are very frequent and with largequantities, often prevailing over cereals.There are three main types ofarchaeobotanical findings of pulses in earlyagricultural sites of the Ararat valley: charredseeds from soft archaeological sediments(soil), the impressions of pods valves andsometimes seeds in the clay-made walls ofdwellings and structures. Charred seeds fromarchaeological sediments are generallyinterpreted as remains of food, andimpressions of pods valves are remains ofcrop threshing waste used as temperingmaterial in building clay.

Some decline in pulses cultivation isrecorded for the Late Chalcolithicperiod, 43rd-33rd centuries B.C. Findings ofpulses (pea, lentil and grass pea) arerepresented with charred seeds and they aremuch less than the cereals remains.

Cultivation of pulses has not been acontinuous practice in the South Caucasus.There are practically no records of pulses forthe entire Bronze Age through the EarlyIron Age period, after the second half of 4thto the beginning of 1st millennia cal BC(Table 1), when agriculture in the territory ofArmenia appears monotonic and specializedon cereals cultivation. Similar tendency wasrecorded in the other parts of the Old Worldas well (3, 4), but in the territory of Armeniaand in general in the South Caucasus EarlyBronze Age agriculture seems to be basedonly on cereals cultivation.

Starting from Middle Iron Age (VanKingdom / Urartu), 9th-6th centuriesBC, the cultivation of a series of pulses re-started, including lentil, bittervetch, pea, chickpea and faba bean. Oftenhoards of pulses are found from the VanKingdom settlements (1, 2).

References(1) Gandilyan PA (1998) Archaeobotanicalevidence for evolution of cultivated wheat and barley in Armenia. In: Damania AB, ValkounJ, Willcox G, Qualset CO (eds) Origins of Agriculture and Crop Domestication, ICARDA, Aleppo, Syria, 280-285(2) Hovsepyan RA (2009) Field crops and common weeds at the territory of Armenia in Neolithic – Iron Age periods. PhD thesis. Scientific center of Agriculture and Plant protection MA RA, Yerevan (unpublished)(3) Riehl S (2009) Archaeobotanical evidence for the interrelationship of agricultural decision-making and climate change in the ancient Near East. Quart Int 197:93-114(4) Zohary D, Hopf M, Weiss E (2012) Domestication of Plants in the Old World. Oxford University Press, New York

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Figure 1. Neolithic lentil and bitter vetch from the territory of Armenia: impression of lentil pod left valve (Masis blur, T.M10/1, L332, F9) in building clay (left); charred seeds of lentil (right, above) and bitter vetch (right, below), Aknashen, T5, UF10b, F16), scale size 2 mm

Table 1. Summary on cultivated pulses presence/absence in archaeological periods in the territory of Armenia

Period Neolithic Chalcolithic Early Bronze AgeMiddle

Bronze AgeLate Bronze Age & Early Iron Age

Middle Iron Age(Van Kingdom /

Urartu)

Sites examined (2)

Akn

ashe

n, M

asis

Blur

, Ara

tash

en

God

edzo

r, A

reni

-1, G

etah

ovit-

2

Geg

haro

t, A

raga

tsi-b

erd,

Mar

gaho

vit,

Loru

t, Ts

aghk

asar

-1, A

para

n III

, V

oske

vaz

(Akh

tam

ir), S

heng

avit,

Sot

k-2,

Nor

abak

-1, A

ygev

an

Ner

kin

Nav

er, A

ghav

natu

n, A

ygev

an,

Shag

hat I

Geg

haro

t, A

raga

tsi-b

erd,

Ts

aghk

ahov

it, H

orom

, Hna

berd

, Te

yshe

bain

i, Sh

agha

t III

, Met

sam

or,

Uyt

s (V

P sit

e 21

), Ju

jeva

n

Hor

om, Y

enok

avan

-2, D

vin,

Ara

mus

, A

rgish

tikhi

nili,

Hor

om, K

arm

ir Bl

ur, S

ev-

sev

kare

ri bl

ur, T

sagh

kaho

vit,

Shag

hat

1

Cereals (Poaceae) + + + + + +

Pulses (Fabaceae) + + +

Lentil (Lens culinaris ssp. microsperma) + + +

Bitter vetch (Vicia ervilia) + +

Pea (Pisum sativum, Pisum spp.) + + +

Chickpea (Cicer arietinum) +

Faba bean (Vicia faba) +

Grass pea (Lathyrus sativus) +

Vetches (Vicia spp., wild?) + + + + + +

10

Molecular analysis of ancient DNA isolated from charred pea (Pisum sativum) seeds found at an Early Iron Age settlement in southeast Serbiaby Petr SMÝKAL1*, Aleksandar MEDOVIĆ2, Živko JOVANOVIĆ3, Nemanja STANISAVLJEVIĆ3, BojanBojan ZLATKOVIĆ4, Branko ĆUPINA5, Vuk ĐORĐEVIĆ6 and Aleksandar MIKIĆ6

Abstract: Archaeobotanically, the bulk ofthe 2,572 charred pea (P. sativum) seedsrecovered at the 12th century BC settlementHissar, near the modern town of Leskovacin southeastern Serbia, belongs to thecultivated pea (P. sativum L. subsp. sativum).As wild or semi-wild pea species, such as P.sativum subsp. elatius still grow in the area, itcould be hypothesised that such seeds couldbe possibly collected. To better identify theorigin of sample, we subjected two samplesto molecular analysis using informativechloroplast DNA sequences. In addition tophylogenetically informative singlepolymorphism sites (SNPs) providingrelationship to extant pea forms andspecies, there were also additionalsubstitutions likely attributed to damage ofDNA. On the basis of these moleculardata, we conclude that the material of thestudy was not so much wild pea, but ratheran early domesticated pea. This is the firstreport of successful aDNA extraction fromany legume species so far.Key words: ancient DNA, charredseeds, legumes, paleogenetics, pea

_________________________________________________________________________________________________________

1Palacký University in Olomouc, Faculty of Science, Department of Botany, Olomouc, Czech Republic ([email protected])2Museum of Vojvodina, Novi Sad, Serbia3University of Belgrade, Institute of Molecular Genetics and Genetic Engineering, Plant Molecular Biology Lab, Belgrade, Serbia4University of Niš, Faculty of Sciences and Mathematics, Department of Biology and Ecology, Niš, Serbia5University of Novi Sad, Faculty of Agriculture, Department of Field and Vegetable Crops, Novi Sad, Serbia6Institute of Field and Vegetable Crops, Novi Sad, Serbia

Although pea (Pisum sativum L.) is one ofthe first crops cultivated by man, a lucky findof 2,572 pea seeds in only one sample fromthe 12th century BC settlement Hissar, nearthe modern town of Leskovac insoutheastern Serbia, represents a uniqueexample in the archaeobotany of South EastEurope. The pea from Hissar was a distinctcrop, stored separately from other crops (3).Archaeobotanically, the bulk of pearecovered at Hissar belongs to the cultivatedpea (P. sativum L. subsp. sativum). Severalmorphological characteristics indicate this:smooth surface of the seed coat, “coffee-bean-shaped” hilum, broad ellipsoid seedshape and small size range differencebetween seeds and high weight of charredseeds. On the other hand, there existed anuncertainty about the determination peaseeds lacking testa and hillm in the largestportion of the sample.

As wild or semi-wild pea species, such as‘tall’ pea (P. sativum subsp. elatius (Steven exM. Bieb.) Asch. & Graebn.), still grow in thearea (6) it could be hypothesized that suchseeds could be possibly collected by theHissar population. The cultivation of solewild, ‘tall’ pea is highly unprofitable: high netyield loss due to poor establishment causedby wild-type low germination rates and poddehiscence (1).

To better identify the genetic origin of theHissar charred pea seeds, we subjected twosamples to molecular analysis usinginformative chloroplast DNA sequences (4).The four selected chloroplast DNAloci, trnSG, trnK, matK and rbcL, amplified insix fragments of between 128 bp and 340 bpwith a total length of 1,329 bp weresuccessfully recovered in order to distinguishbetween the cultivated and wild gatheredpea. In addition to the phylogeneticallyinformative single polymorphism sites(SNPs) providing relationship to extant peaforms and species, there were also additionalsubstitutions likely attributed to damagingDNA. Since the majority of these 16substitutions were of the type 2 transitions, itsupports the evidence of analysis of genuineancient pea DNA. This results from thedeamination of cytosine (and 5-methylcytosine) to uracil (and thymine), shown tobe associated with post-mortem damage (2).

On the basis of the obtained moleculardata (Fig. 1), we may conclude that thematerial of the study was not so much wildpea, but rather an early domesticated pea.Consequently, based on a combination ofmorphological and molecular data, we mayalso consider the material an earlydomesticated pea. We speculate that theEarly Iron Age pea from Hissar could havecoloured flower and pigmented testa, similarto field pea (P. sativum subsp. sativum var.arvense (L.) Poir.), today used mostly forforage, and possibly of winter type (5).

It may be said that our results are the firstreport of a successful extraction of ancientDNA (aDNA) from any legume speciesso far (Fig. 2).

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AcknowledgmentsThis work was supported by the projects 173005, 173030, 31016 and 31024 of the Ministry of Education and Science of the Republic of Serbia, IGAPrF-2012-001 of the Grant Agency of Palacký University and ERA-168 SEELEGUMES within the EU programme SEE-ERA.NET Plus and under the auspices of the EU Seventh Framework Programme (FP7).

References(1) Abbo S, Rachamim E, Zehavi Y, Zezak I, Lev-Yadun S, Gopher A (2011) Experimental growing of wild pea in Israel and its bearing on Near Eastern plant domestication. Ann Bot 107:1399-1404(2) Binladen J, Wiuf C, Gilbert MTP, Bunce M, Barnett R, Larson G, Greenwood AD, Haile J, Ho SYW, Hansen AJ, Willerslev E (2006) Assessing the fidelity of ancient DNA sequences amplified from nuclear genes. Genet 172:733-741

(3) Medović A (2005) Plant husbandry of fortified settlement Hisar near Leskovac Southern Serbia (ca. 1350-1000 bc). Leskov Zb 45:201-209)(4) Smýkal P, Kenicer G, Flavell AJ, CoranderJ, Kosterin O, Redden RJ, Ford R, Coyne CJ, Maxted N, Ambrose MJ, Ellis THN (2011) Phylogeny, phylogeography and genetic diversity of the Pisum genus. Plant Genet Resour 9:4-18(5) Smýkal P, Jovanović Ž, Stanisavljević N, Zlatković B, Ćupina B, Đorđević V, Mikić A, Medović A (2014) A comparative study ofancient DNA isolated from charred pea (Pisum sativum L.) seeds from an Early Iron Age settlement in southeast Serbia: inference for peadomestication. Genet Resour Crop Evol DOI 10.1007/s10722-014-0128-z(6) Zlatković B, Mikić A, Smýkal P (2010) Distribution and new records of Pisum sativumsubsp. elatius in Serbia. Pisum Genet 42:15-18

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Figure 1. The phylogenetic position of the Early Iron Age pea from Hisar and its relationship with the other Pisum taxa

Figure 2. Some of the authors after the press conference announcing the first known successful extraction of ancient DNA from charred legume seeds and a series of expeditions in search for Pisum elatius in southeastern Serbia, Museum of Vojvodina Novi Sad, Serbia, late May 2011

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Legumes diversity in the gorges of Carpathian-Balkan Mountain arc in Serbiaby Bojan ZLATKOVIĆ1*, Stefan BOGOSAVLJEVIĆ2, Aleksandar MIKIĆ3 and Branko ĆUPINA4

Abstract: According to its taxonomicaldiversity, legume family is one of mostrepresented families in the rich floras ofSerbia and Balkan Peninsula. Wild-growingrelatives of cultivated species in the flora ofthe gorges of mentioned areas may representa valuable source of desirable traits for theintrogression into cultivated species, such asthe tolerance to abiotic and biotic stress is ofspecial relevance.Key words: Carpathian-BalkanMountains, floristic analysis, gorges,Leguminosae, Serbia

IntroductionThe limestone gorges and canyons of the

Balkan Peninsula are classified as one of themost significant, realistic and potentialcentres of the floristic diversity in Serbia (7).These regions are characterised by a peculiarfloristic diversity and diversehabitats, enabling the survival of the plantswith different ecology, origin and age. Thegorges and canyons play the role of refugiaand therefore have a greatrichness, originality and antiquity of theirfloras (4). Numerous botanical researchesfrom the middle of the last century untiltoday have significantly contributed to theknowledge on their flora and vegetation.However, the specific aspect of the richnessand taxonomic variability of the familyLeguminosae in their flora have not beenstudied yet to the satisfactory extent.

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1University of Niš, Faculty of Sciences and Mathematics, Department of Biology and Ecology, Niš, Serbia ([email protected])2"Filly Farm Pharmacy", Health Institution, Niš, Serbia 3Institute of Field and Vegetable Crops, Novi Sad, Serbia4University of Novi Sad, Faculty of Agriculture, Department of Field and Vegetable Crops, Novi Sad, Serbia

This paper aims at determining thediversity fo the family Leguminosae in thefloras of the seven most important gorges ofthe Carpathian-Balkan Mountainarc, including their comparative analysis (Fig.1). The checklists of the Leguminosae taxarelated to their flora have been extractedfrom the available botanical literature(1, 2, 3, 5, 6, 12) and the in situobservations, while the data on the familyLeguminosae in the whole flora of Serbiawere obtained from the modern literaryresources (8).

Legumes in gorges of eastern and northeast Serbia

The flora of the predominantly limestonegorges and canyons of the southern part ofthe Carpathian-Balkan Mountains, spreadingthrough eastern and northeast Serbia, isrelatively rich in the Leguminosae speciesand is characterised by rare and endemictaxa. Here is present 135 species andsubspecies of Leguminosae in total, being51% in comparison to whole Serbia, with266 taxa.

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Figure 1. Geographic position of examined gorges of the Carpathian-Balkan arc in Serbia

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Out of 33 genera in total in the flora of thecountry, there is 24 legume ones (73%). In ataxonomic sense, the richest are the generaTrifolium L. (31 taxa), Lathyrus L. and Vicia L.(18 taxa), Cytisus Desf. (13 taxa), Medicago L.(10 taxa) and Astragalus L. (8 taxa). Most ofthese genera comprise economicallyimportant crops, thus making the species ofthe examined gorges and canyons potentiallyuseful in applied genetics and plant breeding.The richest in the Leguminosae species is theSićevo gorge, with 102 taxa, followed byĐerdap, with 70 taxa, and SvrljiškiTimok, with 50 taxa. The smallest number isfound in the smaller gorges, such as Grza (23taxa), Resava (20 taxa), Vratna (20 taxa) andMiljkovac (18 taxa). The Sićevo gorge is therichest one if the indexes of floristic diversityof Leguminosae are compared, expressed asa ratio between the number of species andthe area of the location (LogS(L) / LogA). Itis followed by Svrljiški Timok, while theĐerdap gorge, despite having the largestarea, is the third one (Table 1). The gorgeswith the most original and charactersticLeguminosae representatives are Sićevo, with37, Đerdap, with 15, and Resava, with 4 taxa.

As the southernmost of all seven examinedgorges, Sićevo is dominated by theMediterranean species, with 37%, followedby Eurasian, with 22%, and Pontic andCentral European, with 13% each. InĐerdap, there is 26% Mediterranean, 21%Eurasian and 20% and Central Europeanlegume taxa. In the Svrljiški Timokgorge, the Eurasian taxa are prevalent (30%)over the Mediterranean (26%) and somewhathigher proportion of the Pontic species(18%) than in other gorges. Among thePontic species of this gorge, those such asLathyrus pallescens (M. Bieb.) K. Koch andVicia sparsiflora Ten. are of specific interest.The other, smaller, gorges of eastern Serbiaare characterised by an increased number ofthe cosmopolitan species. Of a peculiarimportance are so-called differentialspecies, that is, the taxa that are present inonly one location. In Sićevo, these are theMediterranean taxa, such as Lens nigricans (M.Bieb.) Godr., Pisum sativum subsp. elatius(Steven ex M. Bieb.) Asch. &Graebn.), Trifolium micranthum Viv., T. scabrumL. or T. vesiculosum Savi, that are importantcrop wild relatives and gene pools ofdesirable traits that may be introgressed intothe cultivated species. In Đerdap, thedifferential species are Laburnum anagyroidesMedik., Lathyrus inconspicuus L. and Trifoliumpatens Schreb.

The limestone gorges and canyons, beingthe refugia of flora and vegetation, areextremely important for preserving thebiodiversity, especially for the speciesrequiring a stable environment, as well as forthose with a narrow distribution andendemic taxa (8). The local Leguminosaeendemics are not present in the gorges andcanyons of Carpathian-Balkan Mountains inSerbia, but there may be found the Balkanendemites, that are, the endemics notrecorded out of the Balkan Peninsula (9, 11).

Out of 492 Balkan endemic taxa, typicalfor the floras of Central Serbia and Kosovo(10), 26 belongs to the family ofLeguminosae (5.3%). In the gorges andcanyons of Carpathian-Balkan Mountains, 4Balkan endemic taxa were recorded, namelyCytisus jankae Velen., Genista subcapitataPančić, Trifolium dalmaticum Vis. and T.medium L. subsp. balcanicum Velen. Thegorges of Sićevo and Đerdap have 3 Balkanendemites each, Svrljiški Timok 2, whileMiljkovac has 1 (Fig. 3).

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AreaA

(km2)Number of

taxa

Number of legume taxa

Share of total flora

(%)

LogS / LogA

LogS(L) /LogA

Đerdap 603.08 1013 70 6.91 0.3423 0.2101

Grza 49.40 340 23 6.76 0.3290 0.1770

Miljkovac 27.50 330 18 5.45 0.3385 0.1687

Resava 69.50 297 20 6.73 0.3153 0.1659

Sićevo 100.00 1275 102 8.00 0.3882 0.2511

Svrljiški Timok 66.20 689 50 7.24 0.3629 0.2172

Vratna 4.90 310 20 6.45 0.3724 0.1945

Serbia 88631.00 3662 266 7.26 0.3256 0.2215

Table 1. General (LogS / LogA) and Leguminosae (LogS(L) / LogA) diversity among the gorges of Carpathian-Balkan arc in Serbia; S - number of taxa, A - area, L - Leguminosae species and subspecies

Figure 2. Cluster diagram of taxonomic similarity of Leguminosae flora in the gorges of Carpathian-Balkan arc in Serbia

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ConclusionThe legume flora of limestone gorges and

canyons of Carpathian-Balkan Mountain arcin Serbia, are comparatively rich in nativelegume species, representing an importantgenetic and breeding genepools for grain andforage legume crops. The analysisemphasizes the gorges in thesouth, floristically impacted by the Ponticand Mediterranean regions, as the centres ofbiodiversity for Leguminosae and the flora asa whole. A specific value of the examinedplant groups is the presence of the Balkanendemic species, stressing the importance ofgorges and canyons in preserving localflora.

Acknowledgements The Ministry of Education, Science and Technological Development of the Republic of Serbia (grant 173030) supported this research.

References(1) Bogosavljević S, Zlatković B, Ranđelović V (2007) Flora klisure Svrljiškog Timoka. In: Ranđelović V (ed) Proceedings, 9th Symposium on Flora of Southeastern Serbia and NeighbouringRegions. Niš, Serbia, 1-3 September 2007, 41-54

(2) Mijatović M, Karadžić B, Jarić S, PopovićZ, Pavlović P, Mitrović M, Đurđević L (2007) Contribution to knowledge of the vascular flora of the Resava gorge, Eastern Serbia. Arch Biol Sci55:75-80(3) Miljković M, Ranđelović N, Ranđelović V (2012) Phytogeographical analysis of the flora of Miljkovačka gorge in Eastern Serbia. Biol Nyssana3:2:77-90(4) Mišić V (1981) Šumska vegetacija klisura ikanjona istočne Srbije. Institut za biološkaistraživanja Siniša Stanković, Belgrade(5) Petrić I, Stojanović V, Lazarević P, PećinarI, Đorđević V (2010) Floristic characteristics of the area of NP "Đerdap" and its immediate surrondings. Prot Nat 61:35-59(6) Ranđelović N, Jeremić Ž, AvramovićD, Petrović D (2003) Flora i vegetacija zaštićenogprirodnog dobra Vratna kraj Negotina. Proeedings, 11th Scientific and ProfessionalConference with International Participation on Natural Resource and Environmental Protection& 16th Days of Preventive Medicine of the TimokRegion with International Participation. DonjiMilanovac, Serbia, 2-4 June 2003, 27-31(7) Stevanović V, Jovanović S, Lakušić D, NiketićM (1995) Diverzitet vaskularne flore Jugoslavije sapregledom vrsta od međunarodnog značaja. In: Stevanović V, Vasić V (eds) BiodiverzitetJugoslavije sa pregledom vrsta od međunarodnogznačaja. Ecolibri, Belgrade, Serbia / Faculty of Biology, Belgrade, Serbia, 183-217

(8) Stevanović V, Jovanović S, Lakušić D, NiketićM (1999) Karakteristike i osobenosti flore Srbije injen fitogeografski položaj na Balkanskompoluostrvu i u Evropi. In: Stevanović V (ed) Crvena knjiga flore Srbije 1. Iščezli i krajnjeugroženi taksoni. Ministarstvo za životnu sredinuRepublike Srbije, Biološki fakultet Univerziteta u Beogradu, Zavod za zaštitu prirode RepublikeSrbije. Belgrade, Serbia, 9-18 (9) Strid A, Tan K (1997) Flora Hellenica 1. Koeltz Scientific Books, Königstein(10) Tomović G, Niketić M, LakušićD, Ranđelović V, Stevanović V (2014) Balkan endemic plants in Central Serbia and Kosovo regions: distribution patterns, ecological characteristics, and centres of diversity. Bot JLinnean Soc 176:173-202(11) Turrill WB (1929) The Plant Life of the Balkan Peninsula. A Phytogeographical Study. Clarendon Press, Oxford(12) Zajić V, Lakušić D (2004) Contribution to the flora of the Grza river gorge (E. Serbia). Proceedings, 2nd Congress of Ecological of the Republic of Macedonia with International Participation. Ohrid, FYR of Macedonia, 25-29 October 2003, 415-422

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Figure 3. The Balkan endemic and rare Leguminosae species in the gorges of Carpathian-Balkan arc in Serbia: Cytisus jankae(left, above), Genista subcapitata (left, below) and Ononis pusilla (middle, above) from Sićevo, Trifolium dalmaticum from Miljkovac (middle below) and Trigonella gladiata from Svrljiški Timok (right)

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Pasture and meadow legumes in Serbiaby Aleksandar SIMIĆ and Savo VUČKOVIĆ

Abstract: Perennial legumes arecharacterized with high content of nutritivesubstances, especially protein and representthe most important protein source inlivestock nutrition. Livestock feed obtainedfrom these plant species can be used inseveral ways, from grazing as most efficientand economical way, to preparation of hayand high quality silages and haylages. Thelegumes could provide forage for livestockand help improve soil fertility and pestmanagement, because they fix nitrogen overan extended portion of the year. Thedistribution of good-quality species oflegumes in phytocenoses of Serbiangrasslands is low, as opposed to the highshare of other plants. State of biodiversity ofgrasslands and overall in Serbia is still onhigh level.Key words:flora, legumes, meadow, pasture, Serbia

IntroductionGrain and forage legumes are grown on

some 180 million ha or from 12% to 15% ofthe global arable area (2). Forage legumeshave been the foundation for dairy and meatproduction for centuries. When properlymanaged, they are rich sources ofprotein, fiber and energy. Even in intensiveanimal and milk production, where graincrops are major feed sources, forage legumesare required to maintain animal health.Alfalfa (Medicago sativa L.) is the prevalentforage legume in temperate climates, whileother important temperate pasture speciesinclude clovers (Trifolium spp.), birdsfoottrefoil (Lotus corniculatus L.), and vetches(Vicia spp.).

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University of Belgrade, Faculty of Agriculture, Zemun, Serbia ([email protected])

Legumes are the major element ofgrassland ecosystems on which the foragequality depends. Grazing tolerance andpersistence of the legume components in thepastures are closely related to mechanism ofself-maintenance of the legumes in thepastures. The inclusion of a nitrogen-fixinglegume component in the sward correspondsto the principles of sustainable agricultureand extensive agricultural production (3).Тhey also have a great importance due totheir contribution to an even distribution ofthe yield produced by a pasture by seasons.These species will often naturally reseedthemselves and can be an important, high-quality component of summer pastures. Insoutheastern Europe, the growth of legumesis seriously limited by the ability of eachspecies to grow during usually cold winters.

Role and potential of legumes in the Serbian grasslands

It is estimated that the permanentgrasslands occupy approximately 830,000 hain Serbia (5). Perennial legumes in thecountry are grown as sole crops on morethan 350,000 ha. Economically, the mostimportant and most present are alfalfa, redclover (Trifolium pratense L.), birdsfoot trefoiland white clover (Trifolium repensL.), although during the last decade sainfoin(Onobrychis viciifolia Scop.) and alsike clover(Trifolium hybridum L.) have become veryimportant (6).

Alfalfa, with its well-developed and deeproot system, enables overcoming dryconditions and even in the years with verylow precipitations it produces satisfactoryyields. The legume possessing the highestgrazing tolerance in Serbia is white clover.

Apart from being an important source ofgood-quality fodder, natural grasslands arealso significant in terms of soil protectionfrom erosion and biodiversity or gene fundpreservation. They are also of an exceptionalimportance for the development of livestockproduction in the upland regions since theyoften represent the main or the only sourceof livestock feed. Presently, many naturalmeadows and pastures are in rather badcondition and are being increasinglysubjected to various degradation processes.This is due to the absence of meliorationmeasures, primarily fertilisation, but also dueto unfavourable environmental conditionsand improper exploitation. There, thedistribution of legumes ranges from 6.73%to 34.12%, depending on plant nutrition (1).The grasslands with a higher share oflegumes and lower percentage of otherplants are characterised by a higher crudeprotein content.

In the Serbian pastures, perennial andannual legumes play a special role as apasture components because of their highquality, production and ability to fixatmospheric nitrogen (Fig. 1). On the otherhand, the problems are the lack ofleguminous species, their low share and thelongevity, especially in perenniallegumes, since it limits a successful forageproduction. Widely adapted forage legumesare increasingly important, but there aresome constraints: alfalfa, in Serbia mainlyused for hay production, does not tolerateacid soils, that is, pH values lower than6.2, red clover has a short life span thanmost other pasture legume species, birdsfoottrefoil possesses a very weak competitiveability, while white clover has a low-growthhabit and is associated with a low dry matteryield (4). When used as freshforage, legumes, except birdsfoottrefoil, may cause bloat.

Generally, the most important foragelegumes on the Balkan Peninsula belong togenus Trifolium L., Medicago L:, Lathyrus L.and Vicia L.. Clovers and alfalfa are commonfield crops, but vetches and vetchlings(Lathyrus spp.), especially annual ones, havenot been tested adequately in pastures so far.

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Annual legumesRecent examinations of natural pastures in

Serbia are focused to mark natural grasslandsrich in annual legumes and determiningspecies suitable for livestock. The emphasisis expected to shift from traditional self-regenerating species, such as alfalfa andclovers, to new species that are adapted toshort periods, namely 1 or 2 years, of pasturewhich can be used in between extendedcropping phases or phase pastures.

Naturalized ecotypes of annual pasturelegumes have been sources of well-adaptedcommercial cultivars. Annual legumesrepresent one of the most significant cropsin the global agriculture today, with aprominent place in both animal nutrition andanimal feeding. The conservation of annualpasture legumes seeds has two importantpurposes: 1) the long term preservation ofgermplasm and 2) the availability of a basicmaterial to obtain new varieties well-adaptedto specific soil and climatic conditions.

One of the main goals of the programmesrelated to genetic resources of annual foragelegumes is the conservation of a remarkablegenetic variability of these crops. They arecarried out in the form of establishing andmaintaining collections of accessions ofdiverse origin, with an emphasis on localpopulations and aimed at using in pastureimprovement programmes. One of maingoals is to find and record the hotspots, richin annual legumes important for pastures andhay production. These are biogeographicregions with a rich biodiversity endangeredby the human activities.

There is a lot of identified annual vetchesin the central Balkans. The territory of Serbiais a reservoir of the Vicia biodiversity. Themost widespread vetches are Vicia sativaL., V. hirsuta (L.) Gray, V. tetrasperma (L.)Schreb., V. villosa Roth, V. grandifloraScop., V. lutea L. and V. narbonensis L..Among vetchlings, the most widespread areLathyrus sativus L., L. aphaca L., L. hirsutus L.and L. cicera L.. All species are native toEurope, while some are typical for SouthEurope. L. cicera is grown mainly in SouthEurope and the Mediterranean for forageand grain production. Also, it is identified inthe flora of Serbia. L. cicera may be apromising species in the Balkanenvironments, regarding its use as amultipurpose crop for feed grain in cattleand sheep, fodder, hay and green manure innumerous countries, such as Australia. Incontrast to L. sativus, the grain of L. ciceracontains low amounts of ODAP, while otherplant parts have even more insignificantconcentrations of this neurotoxin.

Grazing may the main use of annualvetches and vetchlings in pastures, but theyare easily damaged by trampling. There is ageneral recommendation not to graze in latespring when the pods are developed.

The collected seeds and herbaria of annuallegumes could be a source for conservationof these species in the form of collections ofaccessions of diverse origin. It is possible touse these local genetic resources for futureforage crop breeeding programmes.

AcknowldgementsProject TR-31016 of the Ministry of Education, Science and Technological Development of the Republic of Serbia.

References(1) Đurić M, Milić V, Ćurčić S, Veljković B (2007) Yield and quality of natural grassland fodder in the Moravički district [Serbia]. Acta Agric Serb 12:61-68(2) Graham PH, Vance CP (2003) Legumes: importance and constraints to greater use. Plant Physiol 131:872-877(3) Naydenova G, Hristova T, Aleksiev Y (2013) Objectives and approaches in the breeding of perennial legumes for use in temporary pasturelands. Biotechnol Anim Husb 29:233-250(4) Simić A, Tomić Z, Vučković S, BijelićZ, Mandić V (2013) Meadow mixtures in Serbia: challenges and perspectives. Proceedings, 10th International Symposium Modern Trends in Livestock Production. Belgrade, Serbia, 2-4 October 2013, 382-398(5) Sokolović D, Radović J, Lugić Z, Simić A (2014) Agriculture, forage crops and grasslands in Serbia: production and breeding. In: SokolovićD, Huyghe C, Radović J (eds) Quantitative TraitsBreeding for Multifunctional Grasslands and Turf.Springer Science+Business Media, Dordrecht, the Netherlands, 17-30(6) Tomić Z, Lugić Z, Radović J, SokolovićD, Nešić Z, Krnjaja V (2007) Perennial legumes and grasses stable source of quality livestock fodder feed. Biotechnol Anim Husb 23:559-572

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Figure 1. Some of the legumes present in the grassland communities in Serbia: (from left to right) Lathyrus hirsutus, Lathyrus nissolia, Vicia hirsuta and Vicia tetrasperma

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Sustainable preservation of indigenous Albanian legumes and their traditional useby Sokrat JANI1*, Agim CANKO2 and Milto HYSO3

Abstract: The diversity of indigenouslegumes in Albania is seriously eroded as aresult of the multiplicity ofenvironmental, political and socio-economicfactors. This paper discusses thedevelopments related to theidentification, collection, evaluation andpreservation of indigenous forms ofleguminous crops, such as common beans(Phaseolus spp.), chickpea (Cicer arietinum) andLathyrus spp., that are threatened byextinction in the Central Albania, as well asthe traditional knowledge on the productionand their use for own consumption of thefarmers in villages. The paper also suggeststhat the sustainable use of plant biodiversityrequires community-driven in situ and on-farm initiatives supported throughknowledge dissemination, marketingefforts, publicity, and cooperation withresearch and governmental structures.Key words:Albania, collection, conservation, exploration, genetic erosion, indigenous legumes

IntroductionAlbania is well-known for its diverse

environment and for high variability incultivated plants. During the long history ofAlbanian agriculture, local farmers carefullyselected plants and seeds for planting anddeveloped numerous farmer-selectedvarieties, which are well-adapted to localconditions. So, it is a country with a widediversity of plant genetic resources. Thereare numerous primitive cultivars, indigenouslandraces and wild species, especially oflegume plants. Primitive cultivars andindigenous landraces are cultivated especiallyin farmers’ orchards, even in the mostremote rural villages (3).

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1Agricultural Technologies Transfer Center , Lushnja, Albania ([email protected])2Agricultural Technologies Transfer Center , Fushe Kruja, Albania3Independent expert on plant genetic resources, Tirana, Albania

Since 1963, the collecting, evaluation andconservation of legume crops has beenorganized by the Agricultural ResearchInstitute (ARI) of Lushnja, with a clear planto select and produce the commercial seedsfor daily needs of agricultural cooperatives.Until the end of 1980s of the lastcentury, 180 accessions of dry beans(Phaseolus vulgaris L.) were collected, including53 landraces and old cultivars. During 1990-2000, another project wasaccomplished, where several collectingmissions took place, especially in the south-eastern regions of the country (1, 4, 5). Afterthis period, the collecting missions werereduced, due to lack of financial funds.

The process of agricultural diversityreduction, which was observed globally in20th century, severely affected the Albanianagriculture. Not only was the plant diversityreduced, but also the level of utilization ofthe indigenous crops.

In order to give our modest contributionto the purpose of preserving indigenouspopulations of legume plants, theSEELEGUMES project was launched.

Main causes of losing the agrobiodiversity in Albania

The Albanian agricultural sector wasdeveloped during the communist period, inthe form of state and collective farms. Thisspecialization had a negative impact onindigenous crop varieties. In a period of 40years, introduced varieties predominated inthese farms while endemic, rare andthreatened varieties were restricted mainly tofamily plots and agricultural research centers.Consequently, information about localvarieties became restricted to the technicalstaff of research centers and the few familiesthat kept indigenous crop varieties (2).

The process of the agrobiodiversity lossbecame more intensive after the collapse ofthe communist state, with the beginning of ademocratic processes in the 1990s, because alot of people started to leave their native landin rural zones and settled in newplaces, especially in urban zones, in order tohave better possibilities of life. Thismovement of rural people was moreemphatic in the mountain zones. TheCentral Albanian zone, which isdistinguished for legume plantsvariations, was also part of this movement. Itwas consequently associated with the loss ofpossibility to cultivate landraces of legumeplants in the future (5).

MethodologyWithin SEELEGUMES, collecting

missions of legume plant in the CentralAlbania were organised. In order tocomplete this project, a working plan wasdesigned, and exploration and collectingmissions were undertaken for legume plantssuch as common bean, lentil (Lens culinarisMedik.), chickpea (Cicer arietinum L.), fababean (Vicia faba L.), vetchlings (Lathyrus spp.)or pea (Pisum sativum L.).

All this process has been conducted bysub-project staff and ATTC, with thecooperation of the Agricultural RegionalServices. Guidelines were followed referringto these main fields: consulting and gatheringof the information about the villages wherewe could be able to get the best variation oflegume plants, exploration and collectionthrough missions in thefield, characterization and evaluation of thecollected populations based on the minimumdescriptors list, recording the localinformation about species growing andtraditional knowledge for the productionpractices and traditional utilization byinterviewing farmers, and other persons.Seed samples were collected only whenfarmers declared their materials have beencultivated for ages in their families withoutexchanging seed or buying it on themarket (7).

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Project outcomesThe project has achieved some

considerable successes. Notably, importantlandraces have been identified, collected, andmorphologically characterized and ademonstration plot has been established. Atthe end of this project, 27 samples ofcommon bean altogether werecollected, including 24 from the Skrapardistrict, two from the Berat district and thelast one from Lushnja (Fig 1). There werealso found 2 samples of vetchlings and onesample of chickpea. So, the total number ofcollected samples was 30. All of the 24samples from the Skrapar district werecollected in 9 villages. Ten samples weretaken only in one village, Liqeth (Table 1).

The conservation of all the collectedpopulations is interesting for the futurebecause of their specialcharacteristics, displaying a good variationamong them. Now we can demonstrate thevariation of the seed, or grain, orkernel, descriptors, especially for theirshape, size, color and type of seed coat. Werealized that the people of the mountainzones prefer colored common bean.So, from the 24 populations of collectedcommon bean, only 9 of them have whiteseed (37.5%). The biggest part populations(26 populations) of common bean are usedas dry grain and only one of them is used asfresh pod. According to the method ofgrowing, 16 populations needed to besupport by a stick, having a climbing planttype, and 11 populations are cultivatedwithout it, being bush type.

All the samples of the legume populationscollected and their field information weredeposited and landraces seed are preservedin the ATTC gene bank. In addition, seedsmaintained in collections will be regularlyrenewed on the seed’s multiplication plot.

The use of landraces with their ability toproduce good harvests without need forexpensive chemical inputs, and theirtolerance to drought, plant pests and diseaseshas significantly reduced the farmers’exposure to risk. Investing is low and thecrops ideally suit to the growing conditions.Most of the farmers use local varieties fortheir own consumption (1, 6). Thereintroduction of the landraces has alsoimproved the nutritional intake of farmerswith the addition of a greater range of pulses.Local farmers appear to prefer the landracesfor their subsistence needs. Although yieldsare lower for the landraces, they attract ahigher price.

The project collected and documentedtraditional knowledge on the uses ofindigenous crops. A recipe book will bepublished and widely distributed to raiseconsumer awareness as soon as possible. Inaddition, dishes prepared from local varietieshave been promoted through food tastingevents and media. As a result, demand forindigenous varieties is growing at the localmarket.

ConclusionsAs mentioned above, the project did not

imply the protection of the entire spectrumof plants important to agriculture that arethreatened by extinction. Otherwise, theproject approach was the development of areplicable model of agricultural biodiversityprotection for a group of the selected localvarieties (legumes) in one region ofAlbania, which could be used as a strategy inother regions or for other crops andvarieties.

Two years of project implementation haveshown that the sustainable use of plantbiodiversity requires community-driven insitu and on-farm initiatives supportedthrough knowledge dissemination, marketingefforts, publicity, and cooperation withresearch and governmental structures. Theapproaches and instruments developed bythe project will be tested in other regions ofAlbania.

Figure 1. The map of the Berat County with its municipalities of Berat and Skrapar, where the grain legume landraces were collected within the SEELEGUMES project

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References(1) Canko A, Celami A, Kashta F, Gjondeda F (2006) Koleksionimi dhe studimi i germoplazmes autoktone të fasules [Collecting, characterization and evaluation of autochthon accessions on common bean]. Bul Shk Bujq 1:17-22(2) Canko A, Jani S, Hyso M(2010) Evaluation of some growth characters in common bean (Phaseolus vulgaris L.). Sci Stud ResBiol 18:70-75(3) Faslia N, Canko A, Jani S, Miho L (2010) Current status of the national grain legumes collection in Albania. Report of a Working Group Meeting on Grain Legumes, Fourth Meeting, Lisbon, Portugal, 16-17 November 2007(4) Hyso M, Canko A (2001) Koleksionimi dhe studimi i popullatave të fasules. Stud Biol 5-6:370-373(5) Jani S, Canko A (2010) Review on genetic resources of grain legumes in Albania. Ratar Povrt47:617-622(6) Jani S, Miho L (2008) The autochthon French bean cultivation technology. Proceedings, National workshop on technology transfer of techniques related to the use of Plant Genetic Resources for Food and Agriculture (PGRFA), Tirana, Albania, 13-14 November 2008(7) Negri V (2009) Fagiolina (Vigna unguiculatasubsp. Unguiculata (L.) Walp.) from Trasimeno Lake (Umbria Region, Italy). European landraces: on-farm conservation, management and use. Bioversity Tech Bull 15:177-183

Table 1. District and villages and bean landraces collected

District VillageNumber of collected landraces

Dry bean or fresh pod Plant type

Dry bean Fresh Climbing Bush

Berat Lapardha 2 2 - 1 1

Skrapar

Gjerbes 5 5 - 4 1

Gjergjove 1 1 - - 1

Kovacanj 1 1 - - 1

Liqeth 10 9 1 7 3

Luadh 1 1 - - 1

Nishove 1 1 - - 1

Potom 2 2 - 2 -

Strore 2 2 - 2 -

Trebel 1 1 - - 1Lushnje Hajdaraj 1 1 - - 13 11 27 26 1 16 11

Figure 2. A visit to a household in the village of Potom in the municipality of Skrapar

Figure 3. Gathering in situ information on the traditional local practices in cultivating grain legumes from the farmers

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Collecting, characterisation and evaluation of ‘tall’ pea (Pisum sativum subsp. elatius) in southeastern Serbiaby Aleksandar MIKIĆ1*, Branko ĆUPINA2, Bojan ZLATKOVIĆ3, Petr SMÝKAL4, Svetlana ANTANASOVIĆ2, Vuk ĐORĐEVIĆ1, Aleksandar MEDOVIĆ5, Đorđe KRSTIĆ1 and Vojislav MIHAILOVIĆ1

Abstract: In 2011 and 2012, severalexpedition in the upper flow of the riverPčinja, southeastern Serbia, rediscovered acomplex wild population of Pisum elatius inthe forest on the slopes of the MountKozjak An in situ analysis of his pea taxonshowed a considerable potential for forageand grain production of very good quality. Ithas also shown a good tolerance to bothlength and intensity of low temperatures. P.elatius is regarded as able to bring more lightonto the pea crop domestication.Key words: crop wild relatives, in situevaluation, Pčinja, pea, Pisum sativum subsp.elatius, southeastern Serbia

IntroductionSo-called ‘tall’ pea or simply Pisum elatius (P.

sativum L. subsp. elatius (Steven ex M. Bieb.)Asch. & Graebn.) is regarded as a directprogenitor of cultivated pea (5).Ecologically, P. elatius is natively distributedinAlbania, Algeria, Azerbaijan, Bulgaria, Croatia, Cyprus, Egypt, France, FYR ofMacedonia, Georgia, Greece, Iran, Israel, Italy, Lebanon, Libya, Montenegro, Morocco, Portugal, Russia, Romania, Serbia, Spain, Syria,Tunisia, Turkey and the Ukraine (3).

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1Institute of Field and Vegetable Crops, Forage Crops Department, Novi Sad, Serbia([email protected])2University of Novi Sad, Faculty of Agriculture, Department of Field and Vegetable Crops, Novi Sad, Serbia3University of Niš, Faculty of Sciences and Mathematics, Department of Biology and Ecology, Niš, Serbia4Palacký University at Olomouc, Faculty of Sciences, Department of Botany, Olomouc, Czech Republic5Museum of Vojvodina, Novi Sad, Serbia

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Figure 1. The area in southeastern Serbia where a wild population of Pisum elatius (green-outlined elipse) was collected in 2011 and 2012


Figure 2. The monastery of Saint Prohor of Pčinja, 11th century, with the Mount Kozjak in the background and the locations where wild populations of P. elatius were discivered in 2009 (greater yellow-outlined elipse) and in 2011 and 2012 (smaller yellow-outlined elipse)

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The first records on P. elatius in the flora ofSerbia were made in the second half of 19thcentury by one of the most eminent Serbianbotanists, Josif Pančić (4). There are alsocurious reports on a white-flowered varietyalbiflorum (2). In 2009, a usually pink-flowered population of P. elatius wasrediscovered in the the upper valley of thePčinja river, one the very border of thebetween Serbia and Former YugoslavRepublic of Macedonia (Fig. 1). There, P.elatius grows in the lower zone of northerlyexposed slopes of the Mount Kozjak, on theleft riverbank and amidst the termophiloussubmediterranean forests and scattered scrubvegetation on siliceous rocky soil (6).

The SEELEGUMES expeditionsFirst expeditions were made in 2011

aiming at collecting samples for the in situevaluation of the P. elatius population fromthe region of upper flow of the river Pčinjaand the monastery of Saint Prohor Pčinjski(Fig. 2), with emphasis on agronomiccharacteristics, such as yield components andchemical composition (1).

The first expedition was carried out on 28May 2011, targeting the same area where P.elatius was rediscovered in 2009. One groupof several plants was found on the edgebetween the foothills of the MountKozjak, between the forest and the meadowin the very vicinity of the Pčinja river and(Fig. 3 and Fig. 4.) Above this group, up thesteep slopes of the Mount Kozjak and in thedense wood of oak and beech trees andshrubs, two more populations of P. elatiuswere identified, with plants either creepingover the rocky ground or climbing up andeven hanging from the trees branches morethan 1 m high. Several plants were detectedon the completely inaccessible slopesbetween two upper and the lowerpopulation. All the plants were in the stagesof full bloom and developing first pods (Fig.5).

The second expedition, performed on 25June 2011, visited the same population. Allits three groups were in full maturity of theoldest pods, with few already shattering theirseeds. Unlike its immature hanging pods ormature pods in P. sativum subsp. sativum, themature ones were erect, following thepeduncle direction, and with the seeds ofirregular shape and seed coat colour (Fig. 6).

Both expeditions resulted in the samples ofall plant organs for analysing agronomiccharacteristics and chemical composition ofboth forage and grain (1).

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Figure 3. The lowest of three groups of the Pisum elatius population from the upper flow of the river Pčinja, southeastern Serbia, late May 2011

Figure 5. Flowers (left) and hanging immature pods (right) in the plants of the Pisum elatiuspopulation from the upper flow of the river Pčinja, southeastern Serbia, late May 2011

Figure 4. Happy authors with the sample of the lowest group of the Pisum elatius population from the upper flow of the river Pčinja, southeastern Serbia, late May 2011

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The analysis of the fresh abovegroundplant biomass in the stages of full bloom anddeveloping pods suggests that this P. elatiusmay be a potential forage crop (Table 1).

The average seed yield of the collected P.elatius population (5.60 g plant-1) could beconsidered high (Table 2). Its harvest indexis also considerably high for a wild relative ofa crop plant (0.38).

The average crude protein content in theP. elatius forage and grain dry matter may beregarded as rather high (Table 3).

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Table 1. In situ forage yield components of the lowest of three groups of the Pisum elatius population from the upper flow of the river Pčinja, southeastern Serbia, late May 2011 (1)

Stages of full bloom and developing first pods

Stem length (cm)

Number of stems

(plant-1)

Stem mass (g plant-1)

Leaf mass (g plant-1)

Plant mass (g plant-1)

Stem proportion

Leafproportion

Minimum 31 1 4.48 7.70 12.18 0.13 0.48

Maximum 192 8 35.14 63.42 98.56 0.52 0.87

Average 68 3.8 11.04 22.44 33.48 0.33 0.67

Table 2. In situ grain yield components of the lowest of three groups of the Pisum elatius population from the upper flow of the river Pčinja, southeastern Serbia, late May 2011 (1)

Stage of full maturity of first pods and grains

Stem length (cm)

Number of stems

(plant-1)

Number of pods

(plant-1)

Number of grains

(plant-1)

Stem mass

(g plant-1)

Leaf mass

(g plant-1)

Pod mass

(g plant-1)

Grainmass

(g plant-1)

Plant mass

(g plant-1)

Harvest index

Minimum 52 1 2 12 2.82 1.28 0.61 2.46 7.17 0.34Maximum 168 7 11 74 14.34 5.25 2.35 15.71 37.65 0.42Average 72 4.1 4.3 27.3 5.52 2.38 1.24 5.60 14.74 0.38

Crude protein

Crude fat

Crude fibre

Neutral detergent

fibre

Acid detergent

fibreLignin

Crude ash

Nitrogen-free

compoundsStages of full bloom and developing first pods

Stem 115 8 483 619 532 136 61 333Leaf 244 25 265 353 320 62 76 391Plant 201 19 337 441 390 86 71 372LSD0.05 32 6 123 45 35 15 6 29

Stage of full maturity of first pods and grainsStem 69 12 528 674 587 128 88 303Leaf 146 41 291 324 307 76 131 390Pod 98 5 437 573 415 93 70 391Grain 343 - 188 242 205 19 - -LSD0.05 67 11 211 69 55 27 33 31

Table 3. In situ nutritive value of the forage and grain dry matter (g kg’1) of the lowest of three groups of the Pisum elatius population from the upper flow of the river Pčinja, southeastern Serbia, late May 2011 (1)

Figure 6. A mature pod, regularly following the peduncle direction (left), and the mature seeds (right) of the Pisum elatius population from the upper flow of the river Pčinja, southeastern Serbia, late June 2011

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The third expedition was done in mid-November 2011, discovering that theshattered seeds produced on the same yeargerminated most likely during October in alarge number, confirming that P. elatius is notnecessarily characterised by a full seeddormancy.

The fourth expedition in late March 2012brought forth several importantfindings, with an emphasis on the wintersurvival of the P. elatius plants to a greatextent, despite the absolute minimumtemperature of -20 C in January 2012, and agood spring regeneration (Fig. 7) and earlydevelopment of root nodules (Fig. 8).

The fifth and the last expedition to theslopes of the Mount Kozjak was done inJune 2012 (Fig. 9). The collected seeds of theP. elatius population were regenerated andmultiplied for further ex situ research at theExperimental Field of the Institute of Fieldand Vegetable Crops at Rimski Šančevi (Fig.10), as well in an exhibition plot of theArchaeobotanical Garden of the Museum ofVojvodina in Novi Sad in 2011 (Fig. 11).

AcknowledgmentsThis work was supported by the projects 173005, 173030, 31016 and 31024 of the Ministry of Education and Science of the Republic of Serbia, IGA_PrF_2014001 of the Grant Agency of Palacký University and ERA-168 SEELEGUMES within the EU programme SEE-ERA.NET Plus and under the auspices of the EU Seventh Framework Programme (FP7).

References(1) Ćupina B, Zlatković B, Smýkal P, Mikić A, Jajić I, Zeremski-Škorić T, Medović A (2011) In situ evaluation of a Pisum sativum subsp. elatiuspopulation from the valley of the river Pčinja in southeast Serbia. Pisum Genet 43:20-24(2) Kojić M (1972) Grašak (Pisum L). In: Josifović M (ed) Flora SR Srbije IV. Serbian Academy of Sciences and Arts, Belgrade, Serbia, 386-388.(3) Maxted N, Ambrose M (2001) Peas (Pisum L). In: Maxted N, Bennett S (eds) Plant Genetic Resources of Legumes in the Mediterranean. Kluwer, Dordrecht, the Netherlands, 181-190 (4) Pančić J (1874) Flora Kneževine Srbije. Državna štamparija, Belgrade(5) Zeven AC, Zhukovsky PM (1975) Dictionary of Cultivated Plants and Their Centres of Diversity. Centre for Agricultural Publishing and Documentation, Wageningen(6) Zlatković B, Mikić A, Smýkal P (2010) Distribution and new records of Pisum sativumsubsp. elatius in Serbia. Pisum Genet 42:15-17

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Figure 7. In situ comparison of a plant that germinated in the autumn 2011 and survived the winter, with a degraded and withered seed, the new shorter stems and smaller leaves and the longer stems and learger and partially damaged leaves during winter (left one) and a plant that germinated in the spring 2012 (right one), both belonging to the Pisum elatius population from the upper flow of the river Pčinja, southeastern Serbia, late March 2012

Figure 8. In situ root with nodules of a Pisumelatius plant from the population from the upper flow of the river Pčinja, southeastern Serbia, late March 2012

Figure 9. More of the happy authors visiting again one of the upper Pisum elatiuspopulations from the upper flow of the river Pčinja, southeastern Serbia, examining its microenvironment and taking soil samples, early June 2012

Figure 10. Ex situ testing of the Pisum elatiuspopulation from the upper flow of the river Pčinja, southeastern Serbia, at Rimski Šančevi, northerm Serbia, late June 2012

Figure 11. Even more of the happy authors in the Archaeobotanical Garden of the Musem of Vojvodina, Novi Sad, with the Pisum elatiuspopulation from the upper flow of the river Pčinja, southeastern Serbia, in the company of Priapus, Greek god of fertility, April 2013

The richness of the biodiversity in Bosniaand Herzegovina was noted more than 100years ago (1), but the significant organizedand long-term measures to protect thiswealth have never been taken. Bosnia andHerzegovina, that is, the Republic ofSrpska, has favorable agroecologicalconditions, characterized by the abundanceof forage legumes and grasses.

Through the project SEELEGUMES, withthe University of Banja Luka as one of thepartners, a small collection of faba bean(Vicia faba L.) landraces was established.Faba bean in Srpska can be found mainly ineastern Herzegovina and the regions close tothe Adriatic coast (2). These varieties areforused mainly for humanconsumption, while faba bean varieties foranimals are negligible (Fig. 1).

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Reintroduction of faba bean (Vicia faba L.) in the Republic of Srpskaby Branko ĐURIĆ

Abstract: Faba bean in Srpska can be foundmainly in eastern Herzegovina and theregions close to the Adriatic coast. Thesevarieties are forused mainly for humanconsumption. In the cooperation with theInstitute for Field and Vegetable crops fromNovi Sad, Serbia, 13 landraces of fodder fababean were tested for five years in the BanjaLuka region. The results indicate that fababean may be an important annual forage andgrain legume, although it is underrepresentedin the sowing structure and cropping system.Key words: crop reintroduction, fababean, genetic resources, landraces, Republicof Srpska

Social and scientific importance of plantgenetic resources for the wholehumanity, community and every individual ispriceless. Genetic resources have a specialeconomic and breeding importance forbreeding activities for a certain plant species.

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University of Banja Luka, Faculty of Agriculture, Banja Luka, Republic of Srpska, Bosnia and Herzegovina ([email protected])

In the cooperation with the Institute forField and Vegetable Crops from NoviSad, Serbia, 13 landraces of fodder faba beanwere tested for five years in the Banja Lukaregion (Fig. 2). The aim of the experimentwas to investigate the faba bean yield andadaptability of the cultivars in this region.The results indicate that faba bean may be animportant annual forage and grainlegume, although it is underrepresented inthe sowing structure and cropping systems.Due to a number of favorablecharacteristics, a greater presence of fababean in our arable land is recommended.Further step would be the establishment ofdemonstration trials on selected farms in thisregion.

References(1) Beck G (1927) Flora Bosne i Hercegovine ioblasti Novog Pazara. Prirodnjački i matematičkispisi, LXIII, Serbian Royal Academy, Belgrade -Sarajevo(2) Mikić A, Anđelković S, Đurić B, VasićM, Ćupina B, Mihailović V, Duc G, Marget P (2011) Status of the local landraces of faba bean in west Balkans. Grain Legum 56:8

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Figure 2. A trial with local faba bean landraces from Serbia and Srpska, Banja Luka, 2008

Figure 1. The seed of a faba bean landrace from Herzegovina

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Local plant genetic resources of faba bean (Vicia faba) in Bulgariaby Siyka ANGELOVA*, Mariya SABEVA* and Yana GUTEVA

Abstract: The grain legume collection atInstitute of Plant Genetic Resources ”K.Malkov” (IPGR) consists over 5,000accessions. The main task within the projectSEELEGUMES was identifying faba beanlocal landraces, still grown and maintained inthe home gardens and small farms, withabout 100 accessions collected. The collectedaccessions were classified by duration of thegrowing season, plant habit and height, massand color of the seeds and content of crudeprotein. The ex situ conservation of fababean in the gene bank is necessary becausethey are endangered and are an importantpart of the rural cultural heritage of thecountry.Key words: Bulgaria, ex sittuconservation, faba bean, in situ expeditions

Faba bean (Vicia faba L.) is an oldtraditional crop. In Bulgaria, it is grown onsmall areas and primarily for humanconsumption. Faba bean is used to a smallerextent as forage and green manure.

The grain legume collection at Institute ofPlant Genetic Resources ”KonstantinMalkov” (IPGR) consists over 5,000accessions. The faba bean is represented by770 accessions, with only 10-12% ofBulgarian origin.

The main task within the projectSEELEGUMES was collecting faba beanlocal landraces, because they are still grownand maintained in the home gardens andsmall farms almost in the whole country (Fig.1). They were visited and taken an inventoryof over 20 regions, with collecting about 100accessions of local populations, varieties andforms (Fig. 2).

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Institute of Plant Genetic Resources ”K.onstantinMalkov” Sadovo, Bulgaria([email protected], [email protected])

The organization of ten expeditions wasmotivated by a single reason - depopulatingnumerous villages and losing valuable geneticresources.

In our country, the traditional grain legumecrops that are still grown are mostly fababean and common beans (Phaseouls spp.)and, rather rarely, pea (Pisum sativum L.) andchickpea (Cicer arietinum L.). The results ofthe characterization and evaluation show thatthe local faba bean landarces are very diversein phenotype and genotype. The collectedaccessions were classified by duration of thegrowing season, plant habit and height, massand color of the seeds and content of crudeprotein.

The plant height of 2/3 of the studiedaccessions ranges from 81 cm to 100 cm.The rest of them are lower than 80 cm orhigher than 100 cm. Most of the samples areof a medium and large seed size, with 3-4seeds per pod and a size of the pod of 80-90mm /13-15 mm. The content of crudeprotein varies widely, from 26% to 38% ingrain dry matter (Fig. 3). The old traditionalplant materials are grown, conserved andadapted to the conditions of theenvironment with specific regionalcharacteristics. The next stage of theevaluation will include testing the toleranceto various biotic and abiotic stress factors.

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Figure 1. Gathering valuable information on the locally maintained faba bean landraces in one village in Bulgaria

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Local plant genetic resources of faba bean (Vicia faba) in Bulgariaby Siyka ANGELOVA*, Mariya SABEVA* and Yana GUTEVA

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0 5 10 15 20 25 30 35 40

%

26- 28 %

28 -30 %

over 30 %

gro

up

s

Figure 2. Some of the local faba bean landraces from Bulgaria maintained at IPGR, Sadovo, Bulgaria

Figure 3. Local faba bean landraces maintained at IPGR, Sadovo, Bulgaria, grouped after the average grain crude protein content (%)

The project implementation allowed us toenrich the existing collection with localforms, populations and varieties of faba beanthat are still grown in our country. Thecollected database provides the informationon origin, farmer’s name and utilization.

The ex situ conservation of seeds in thegene bank is a necessary preventive measurefor small crops because they are endangeredand are an important part of the ruralcultural heritage of the country. ■

Therefore, a new mission to collect anexpanded number of accessions of C. reticulatumand C. echinospermum P.H. Davis, both of whichcan be crossed with domestic chickpea, wasinitiated in 2013 in SE Turkey. The species’natural habitat is in fallow fields, vineyards, rockylands and oak savannas from 600 to 1,630 m.The modus operandi of the collection missionwas to visit previously known areas of C.reticulatum and C. echinospermum distribution andexpand from there, paying close attention to thehabitatsoutlinedabove.

On many occasions C. reticulatum could notfound in locations where it had previously beencollected (5, 6) indicating that it is decliningspecies of conservation concern. Threats includeroad and dam constructions and the attendantincrease in grazing pressure associated with easyaccess, conversion of pastures and savannas totilled agricultural land, and habitat degradationfrom mining, and are particularly evidentcurrently as Turkey enjoys decade-long economicgrowth. 763 new accessions were collected on asingle plant basis from 41 collection sites, largelyin C. reticulatum (n = 513) and C. echinospermum(n = 169), representing a substantial increase overthe 18 and 10 original accessions in the currentworldcollection(2).

Both C. reticulatum and C. echinospermum possessmany characteristics that are potentially useful inbreeding, such as variable phenology andmorphology (6), extreme temperaturetolerance, and resistance to biotic and abioticstresses (Table 1). Moreover the species harboursignificant genetic variation (8) representing anunder exploited resource for base-broadeningcultivated chickpea in breeding programs (7, 14)and in the development of genome sequencesand mapping (12).

AcknowledgmentsThe first author is greatly indebted to the financialsupport of the Scientific and Technical ResearchCouncil of Turkey (TUBITAK).

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Cicer reticulatum Ladizinsky, progenitor of the cultivated chickpea (C. arietinum L.)by Cengiz TOKER1*, Jens BERGER2, Abdullah KAHRAMAN3, Abdulkadir AYDOGAN4, CananCAN5, Bekir BUKUN6, R. Varma PENMETSA7, Eric J. von WETTBERG6 and Douglas R. COOK7

Abstract: Cicer reticulatum is a member of thegenus Cicer and is considered to be theprogenitor of cultivated chickpea (C. arietinum).During the latest expedition in Turkey, 763 newaccessions were collected on a single plant basisfrom 41 collection sites, namely 513 of C.reticulatum and 169 of C. echinospermum. Both C.reticulatum and C. echinospermum possess manycharacteristics that are potentially useful inbreeding, such as variable phenology andmorphology, extreme temperature tolerance, andresistance to bioticand abiotic stresses.Key words: Cicer arietinum, Cicer reticulatum, cropwildrelatives,genetic resources

Cicer reticulatum Ladizinsky (Fig. 1) is a memberof the genus Cicer L., the tribe CicereaeAlef., subfamily Faboideae orPapilionoideae, family Fabaceae or Leguminosae(9), and is considered to be the progenitor ofcultivated chickpea (C. arietinum L.) (4, 11).Although the oldest archeological seed samplesof C. reticulatum were dated to 7,260 BC fromTell-el-Kerkh, Syria (10), it is an annual speciesendemic to southeastern Turkey (1, 3, 14). Bergeret al. (2) reported only 18 unique accessions of C.reticulatum in national and international genebanksthat have been widely replicated, subsampled andrenamed to create the number of accessionscurrentlyavailable.

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1Akdeniz University, Faculty of Agriculture, Department of Field Crops, Antalya, Turkey ([email protected])2CSIRO, Plant Ind. Wembley, Australia3Harran University, Faculty of Agriculture, Department of Field Crops, Şanlıurfa, Turkey4Field Crops Central Research Institute, Yenimahalle, Ankara, Turkey5Gaziantep University, Faculty of Art and Science, Department of Biology, Gaziantep, Turkey6Department of Biol. Sci. Miami, USA7University of California Davis, Department of Plant Pathology, Davis, USA

References(1) Abbo S, Redden RJ, YadavSS (2007) Utilization of wild relatives. In: YadavSS, Redden B, Chen W, Sharma B (eds) Chickpea Breeding and Management. CAB International, Wallingford, UK, 338-354(2) Berger J, Abbo S, Turner NC (2003) Ecogeography of annual wild Cicerspecies: The poor state of the world collection. Crop Sci43:1076-1090(3) Ladizinsky G (1975) A new Cicerfrom Turkey. Notes R BotGardEdinb34:201-202(4) Ladizinsky G, Adler A (1976) The origin of chickpea (CicerarietinumL.). Euphytica25:211-217(5) OzturkM (2011) Revision of the genus CicerL. in Turkey via morphological, palynological, cytotaxonomical, molecular phylogeneticmethods, and analyses of seed protein and element contents. PhD Thesis. The Graduate School of Natural and applied Science of SelcukUniversity, Konya(6) Roorkiwal M, von Wettberg EJ, UpadhyayaHD, Warschefsky EJ, Rathore A, Varshney RV (2014) Exploringgermplasm diversity to understand the domestication process in chickpea (Cicer) using SNP and DArTmarkers. PloSONE9: e102016(7) Robertson LD, Singh KB, Ocampo B (1995) A Catalog of Annual CicerSpecies. ICARDA, Aleppo(8) Shan F, Clarke H, Plummer J, Yan G, SiddiqueKHM (2005) Geographical patterns of genetic variation in the world collections of wild annual Cicercharacterized by amplified fragment length polymorphisms. TheorAppl Genet 110:381-391(9) Smýkal P, Coyne CJ, Ambrose MJ, MaxtedN, Schaefer H, Blair MW, Berger J, Greene SL, Nelson MN, BesharatN, Vymyslický T, TokerC, SaxenaRK, Roorkiwal M, PandeyMK, HuJ, Li YH, Wang LX, Guo Y, QiuLJ, Redden RJ, Varshney RK (2015) Legume crops phylogeny and genetic diversity for science and breeding. CritRev Plant Sci34:43-104(10) Tanno K, WillcoxG (2006) The origins of cultivation of Cicerarietinum L. and Viciafaba L.: early finds from Tell el-Kerkh, north-west Syria, late 10th millennium B.P. Veg HistArchaeobot 15:197-204(11) TokerC (2009) A note on the evolution of kabuli chickpeas as shown by induced mutations in Cicerreticulatum Ladizinsky. Genet ResourCrop Evol56:7-12(12) TokerC, Uzun B, Ceylan FO, Ikten C (2014) Chickpea. In: PratapA, Kumar J (eds) Alien Gene Transfer in Crop Plants 2, Springer Science+Business Media, Dordrecht, the Netherlands, 121-151(13) Warschefsky EJ, PenmetsaRV, Cook DR, von WettbergEJ (2014) Back to the wilds: tapping evolutionary adaptations for resilient crops through systematic hybridization with crop wild relatives. Am J Bot101:1791-1800(14) Zohary D, HopfM (2000) Domestication of Plants in the Old World. Oxford University Press, New York

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Figure 1. Cicer reticulatum Ladiz. in its natural habitat with leaves, flower and immature pod (left) and mature pod and seed (right)

Stresses Accessions References*

Cold

ILWC 81, ILWC 112, ILWC 113, ILWC 117, ILWC 139, ILWC 140, ILWC 141

Singh et al. 1990, 1994, 1995, 1998

ILWC 81, ILWC 104, ILWC 105, ILWC 106, ILWC 108, ILWC 109, ILWC 110, ILWC 111, ILWC 112, ILWC 113, ILWC 114, ILWC 115, ILWC 116, ILWC 117, ILWC 118, ILWC 120, ILWC 122, ILWC 123, ILWC 124, ILWC 125, ILWC 126, ILWC 127, ILWC 128, ILWC 129, ILWC 130, ILWC 131, ILWC 134, ILWC 135, ILWC 136, ILWC 137, ILWC 139, ILWC 140, ILWC 141, ILWC 142, ILWC 182, ILWC 183, ILWC 184, ILWC 216, ILWC 218, ILWC 219, ILWC 229, ILWC 231, ILWC 233, ILWC 242

Robertson et al. 1995

AWC 600, AWC 601, AWC 602, AWC 603, AWC 604, AWC 605, AWC 606, AWC 607, AWC 608, AWC 609, AWC 610, AWC 611, AWC 612, AWC 613, AWC 614

Toker 2005

ILWC 81, ILWC 106, ILWC 139 Saeed et al. 2010

Drought and heat AWC 605, AWC 616, AWC 620, AWC 625 Canci and Toker 2009

Drought ILWC 36, ILWC 116 Imtiaz et al. 2011

Ascochyta blight

ILWC 113 Singh et al. 1998

ILWC 26, ILWC 130 Infantino et l., 1996

ILWC 104, ILWC 118, ILWC 119, ILWC 139, Collard et al. 2001

ILWC 17+21 Shah et al. 2005

Fusarium wiltILWC 26, ILWC 130 Infantino et l., 1996

ILWC 81, ILWC 112, ILWC 117, ILWC 139, ILWC 140, ILWC 141 Singh et al. 1998

Botrytis gray mold IG 72959, IG 72933, IG 72941 Pande et al., 2006

Crenate broomrape C 553 Rubiales et al. 2004

Cyst nematodeILWC 119 Di Vito et al. 1996

ILWC 81, ILWC 112, ILWC 117, ILWC 139, ILWC 140, ILWC 141 Singh et al. 1998

Table 1. Sources of Cicer reticulatum Ladiz. for resistance to biotic and abiotic stresses

Microsatellite markers, also called SimpleSequence Repeats (SSR), are user-friendlyhighly polymorphic markers suitable forgenetic diversity studies, puritycontrol, differentiation of plantcultivars, elimination of duplicates andselection of appropriate parents based ongenetic distances in breeding programs (1).The genetic diversity of 119 common bean(Phaseolus vulgaris L.) accessions originatingfrom five SEE countries was performedusing 13 SSR markers (2). Analysis included25 accessions from Bosnia andHerzegovina, 18 accessions from Croatia, 28accessions from Macedonia, 30 accessionsfrom Serbia and 18 accessions fromSlovenia. The calculated mean number ofalleles per locus was 9.08. The averagepolymorphic information content over allloci reached value of 0.72. These resultsshowed that the selected set of SSR markersis highly informative and applicable forstudies of common bean genetic diversity(2). In the Principal Component Analysis(PCoA) plot (Fig. 1), the majority ofaccessions were grouped into two distinctclusters, which resemble to Mesoamericanand Andean gene pools. Both clusterscomprised accessions from each of the fivecountries. The accessions from Macedoniaand Serbia were distributed evenly betweenthe two gene pools, while the accessionsfrom Croatia, Bosnia and Slovenia clusteredpredominantly within the Andean gene pool.Additionally, PCoA and Bayesian clusteranalysis indicated the existence of potentialhybrids between the two gene pools. Theanalysis of molecular variance revealed thatsignificant part of accessions from differentcountries is closely related. This study showthat common bean might be introduced intothe territory of former Yugoslavia severalcenturies ago by a common route (2).

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South East European (SEE) autochtonous Phaseolusbean germplasm genetic diversity and reintroduction of traditional landracesby Vladimir MEGLIČ*, Jelka ŠUŠTAR-VOZLIČ and Marko MARAS

Abstract: Genetic diversity of 25 accessionsof common bean (Phaseolus vulgaris L.) fromBosnia and Herzegovina, 18 fromCroatia, 28 from Macedonia, 30 from Serbiaand 18 from Slovenia was assessed with 13Simple Sequence Repeats (SSR) markers.The results showed that selected set of SSRmarkers are highly informative andapplicable for studies of common beangenetic diversity. The gene bank at theAgricultural institute of Slovenia (AIS) holdsa collection of 1,035 bean accessionscollected from various parts of Slovenia inthe last decades. With published informationand description of old varieties and growingtechnologies, their traditional use, the waysof preservation and/or processing as well asrecipes of traditional dishes, we will promotepreservation of traditional knowledge.Key words: common bean, geneticresources, landraces, Phaseolus, Slovenia

The selected regions of Slovenia and otherregions of Southeast Europe (SEE) are stillrich in biodiversity and genetic resources ofgrain legumes constituting a great potentialfor their utilization in breeding programs orfor direct reintroduction back to the farm.Due to changes in climate conditions andagricultural practices there is an impendingrisk of losing valuable genetic diversity. TheSEELEGUMES project helped to ensure along-term conservation of legume plantgenetic resources and to promote theirsustainable utilisation in the SEE region andstrengthened the national efforts.

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Agricultural Institute of Slovenia, Ljubljana, Slovenia ([email protected])

Common bean has been cultivated inSlovenia for centuries, resulting in thedevelopment of numerous landraces, someof which are still grown today. The genebank at the Agricultural Institute of Slovenia(AIS) holds a collection of 1,035 beanaccessions collected from various parts ofSlovenia in the last decades (3, 4). The resultsrevealed that during the centuries ofcommon bean cultivation in Slovenia adiverse collection was formed that should bepreserved for the future (4). With the aim toreintroduce traditional landraces back tocultivation, 16 Češnjevec accessions and 23accessions of landrace Lišček (anothertraditionally grown common bean type) wereselected from the AIS gene bank. Fieldstudies were conducted in three successiveyears; plant growth, resistance to diseasesand pests were evaluated and the yield wasdetermined. The chemical composition ofraw seeds was analysed, which includeddetermination of macro- andmicroelements, dry matter and starchcontent, crude proteins, crude fiber andpolyphenols, and the sensory characteristicswere evaluated. Based on the resultsobtained, three landraces, one Češnjevec andtwo Lišček accessions, were selected for theinscription in the national list of varieties ofSlovenia as conservation varieties (5).

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References(1) Maras M, Šuštar Vozlič J, Kainz W, Meglič V(2013) Genetic diversity and dissemination pathways of common bean in Central Europe. J Am Soc Hortic Sci 138:297-305(2) Meglič V, Pipan B, Šuštar Vozlič J, Maras M, Todorović V, Vasić M, Kratovalieva S, Ibusoska A, Matotan Z, Čupić Tihomir (2013)Genetic diversity of the autochtonous Phaseolusbean germplasm originating from five South east European countries. Abstracts, First Legume Society Conference 2013: A Legume Odyssey, Novi Sad, Serbia, 9-11 May 2013, 29 (3) Meglič V, Šuštar Vozlič J, Verbič J, Ugrinović K, Zemljič A, Čergan Z, Dolničar P, Koron D, Koruza BR, Pilih K, Škof M, Rutar R, Maras M, Pipan B (2013) Genebank of agricultural plants at the Agricultural Institute of Slovenia. Proceedings, Symposium New Challenges in Agronomy, Zreče, Slovenia, 24-25 January 2013, 309-314

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Both data and material of the newlycollected and evaluated accessions will beadded in the database and SEE gene banksthat will serve scientific community, plantbreeders, farmers and hobby gardeners. Thiswill present the information that will enablethe insight in morphological and molecularcharacteristics of the collected/storedmaterial. With published information anddescription of old varieties and growingtechnologies, their traditional use, the waysof preservation and/or processing as well asrecipes of traditional dishes, we will promotepreservation of traditional knowledge.

(4) Šuštar Vozlič J, Maras M, Munda A, Zadražnik T, Meglič V (2012) Variability of common bean accessions in the gene bank of Agricultural institute of Slovenia. Acta Agric Sloven 99: 399-411(5) Šuštar Vozlič J, Maras M, Ugrinović K, Velikonja B, Žnidaršič Š, Pongrac V, Zadražnik T, Meglič V (2014) Reintroduction of traditional Slovenian common bean landraces to facilitate their conservation and use. Abstracts, International Conference on Enchanced Genepool Utilization Capturing Wild Relative and Landrace Diversity for Crop Improvement, Cambridge, United Kingdom, 16-20 June 2014, 126

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Figure 1. A Principal Component Analysis (PCoA ) plot showing genetic relationships among 167 common bean accessions from five South East European countries

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Lignified tissues in vegetative organs of wild-growing Trifolium L. forage speciesby Lana ZORIĆ1, Jadranka LUKOVIĆ1, Dunja KARANOVIĆ1 and Aleksandar MIKIĆ2

Abstract: Wild-growing Trifolium foragespecies are often grazed or cut for hay orfresh forage in their natural habitats. Theproportion of lignified tissues in vegetativeorgans is important parameter that affectsdigestibility, since lignified tissues resistrumen degradation. The examined specieshad the highest proportion of lignifiedtissues in stems and peduncles and thelowest in lamina. They proved to havepreferable proportion of lignified, thick-walled tissues and thin-walled, parenchymatictissues, and were assigned as forages ofpotentially high digestibility and good quality.Key words: anatomy, clovers, foragequality, Trifolium spp.

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1University of Novi Sad, Faculty of Sciences, Department of Biology and Ecology, Novi Sad, Serbia ([email protected])2Institute of Field and Vegetable Crops, Novi Sad, Serbia

The Trifolium L. forage species are amongthose forages that are used in plantedpastures, but also utilized by grazing animalsin their natural habitats or cut for hay orfresh forage, without being deliberately sown(4). Plant breeding programs improveagronomic characteristics of legumeforages, but also their adaptation to theenvironment. Wild relatives of the fewcommercialized Trifolium species occurnaturally and are frequently grazed.Moreover, local populations, which are likelyto be better adapted to local environmentalconditions, can serve as sources of geneticvariation.

Lignified tissues have important role inproviding plant’s resistance, but negativelyaffect their digestibility (2). Tissues with ahigh proportion of thick-walled cells areincompletely digested or resistdegradation, and the reduction of theirproportion is one of the objectives of foragebreeding programs (1). In Trifolium speciessuch tissues are sclerenchymatousparenchyma (present only in stem andpeduncle), vascular tissue andsclerenchyma, the proportion of whichshould be minimized, without negativelyaffecting plant’s resistance and strength (3).Parenchyma tissues are thin-walled, whilstepidermis and collenchyma have cells withthickened, non-lignified cell walls.

Using anatomical method, lightmicroscopy observations and measurementsof cross-sections, the proportions of lignifiedand non-lignified thin-walled tissues weredetermined in plant organs of wild-growingTrifolium forage species (Fig. 1). Stems andpeduncles had the highest proportion oflignified tissues (15.5% - 29.6% and 14.9% -26.3%, respectively), which made theseorgans the most responsible for digestibilitylimitations. Parenchyma tissue was bestdeveloped in lamina (87.7% - 92.4%), whichcould contribute to high leafdigestibility, which is characteristic of legumeforages. The examined species showed highlevel of similarity in examinedparameters, which generally had lowvariability. The most desirable ratio oflignified and non-lignified tissues wasrecorded in T. subterraneum L.

The proportion of lignified tissues inexamined Trifolium species was significantlylower compared to the one obtained forwild-growing Trifolium species that were notrecognized as forages (3). The examinedspecies also had preferable percentage oflignified tissues compared to the percentagerecorded in vegetative organs of cultivatedMedicago truncatula Gaertn. (6), or in stems ofwild-growing Lathyrus L. species (5).According to the results of anatomicalanalysis, the examined Trifolium speciesrepresent wild-growing forages of potentiallyhigh digestibility and good quality.

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References(1) Buxton DR, Redfearn DD (1997) Plant limitations to fiber digestion and utilization. J Nutr 127:814-818(2) Guines F, Julier B, Ecalle C, Huyghe C (2003) Among and within-cultivar variability for histological traits of lucerne (Medicago sativa L.) stem. Euphytica 130:293-301(3) Krstic L, Merkulov Lj, Lukovic J, Boza P (2008) Histological components of Trifolium L. species related to digestive quality of forage. Euphytica 160:277-286(4) Sackville Hamilton R, Huges S, Maxted N (2001) Ex situ conservation of forage legumes. In: Maxted N, Bennett SJ (eds) Plant genetics resources of legumes in the Mediterranean. Kluwer Academic Publishers, Dodrecht, the Netherlands, 263-291 (5) Zoric L, Merkulov Lj, Lukovic J, BozaP, Krstic B (2011) Evaluation of forage quality of Lathyrus L. species based on histological characteristics. Acta Agron Hung 59:47-55 (6) Zoric L, Mikic A, Cupina B, Lukovic J, KrsticDj, Antanasovic S (2014) Digestibility-related histological attributes of vegetative organs of barrel medic (Medicago truncatula Gaertn.) cultivars. Zemdirb Agric 101:257-264

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Figure 1. The percentages of tissues composed of lignified (above) and non-lignified thin-walled cells (below) in different organs of Trifolium forage species

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Economic value of perennial vetchlings (Lathyrus L.) species from the Russian Far Eastby Margarita VISHNYAKOVA*, Alla SOLOVYOVA, Andrey SABITOV, Pavel CHEBUKIN and Marina BURLYAEVA

Abstract: This paper presents the results ofthe estimation of protein amount in greenmass of several Lathyrus species, introducedat VIR collection in 2010-2011 from theterritory of the Russian Far East. Theaccessions have been collected from the vastterritory during the expedition surveys. Theinterpopulation variability of the traits havebeen revealed. Preliminary conclusions aboutthe economic value of these species as forageplants are highlighted.Key words: crude protein content, greenmass, Lathyrus, Russian Far East, vetchlings

The collection of the genus Lathyrusrepresentatives in the N.I. Vavilov All-Russian Research Institute of Plant Industry(VIR) in St. Petersburg, Russia, contains1,835 accessions belonging to 56 species. InRussia, only five species are involved inbreeding, namely L. sativus L., L. sylvestrisL., L. tingitanus L., L. chloranthus L. and L.odoratus L.. The last three species are used asornamental plants.

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N.I. Vavilov All-Russian Research Institute of Plant Industry, St. Petersburg, Russia([email protected])

The increase in the species composition ofthe collection expands the possibility ofdomestication of new plants and theirtransformation into crops. Especiallyinteresting is the potential of perennialvetchling (Lathyrus spp.) species, renown fora high protein content, longevity, early springregrowth, ability to regenerate after severalmowing during one season. Thesespecies, along with perennial vetches (Viciaspp.) could be the components ofintercropping, designed for many years ofuse, especially in the areas with unfavourableand complex surface terrain, where theymust be mowed by hand. They also couldenrich the diversity and increase the qualityof natural pastures (1).

The flora of Siberia and the Far East of theRussian Federation contains at least 8perennial Lathyrus species, including 4endemic (3). During 2010-2011, VIRorganized the expeditions to the regions ofPrimorsky and Khabarovsk in Russia and theHeilongjiang Province in Northeast China.The plant samples of 8 species werecollected during late August andSeptember, when mature pods wereformed, enriching the VIR collection with 7new Lathyrus species.

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Figure 1. Protein content in forage dry mass (%) of the perennial accessions of Lathyrusspecies collected in the Russian Far East during 2010-2011

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5

10

15

20

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The protein content in forage dry mass of the collected accessions

Since the collected accessions arepromising, especially as potential foragecrops, the protein content in green mass wasdetermined by the standard Kjeldahlmethod. The protein content was expressedas a percentage in forage dry weight. Theresults showed interspecific and intraspecific(inter-population) variability of the trait. Thesamples collected in different habitats hadthe following protein content in green mass:L. komarovii Ohwi, from 13.81% to 17.41%;L.humilis (Ser.) Sprengel, from 10.32% to16.93%; L. palustris L., from 15.15% to18.01%; L. japonicus Willd., from 16.18% to20.94%; L. davidii Hance, from 19.92% to21.85%; L. subrotundus Maxim., from 16.57%to 19.00% (Fig. 1).

Previously, we had analyzed this characterin the representatives of some perennialspecies from the collection grown in theexperimental fields in Central Russia (thetownship of Ekaterinino, the region ofTambov). The highest value of the trait(20.7%) was observed in the cultivatedspecies L.sylvestris L. (2).

The data for other species grown with anadvanced level of agronomy in Ekaterininoare comparable with those obtained for thesamples from the Russian Far East, with theadvantage of the latter. The highest proteincontent was observed in two samples of L.davidii, collected in Primorye, with 21.34%and 21.85%.

The next step in assessing the nutritionalvalue of these species should be aimed atdetermining the content of anti-nutritionalsubstances, such as ODAP, as wellas of sugars, chlorophyll A andcarotenoids, beta-carotene and theproportion of organic substances.

ProspectsAt this stage, the study of biological

characters and economic assessment has notbeen carried out for the all Lathyrus speciescollected and introduced in the VIRcollection. However, it is known that someof them, such as L. japonicus, L. davidii, L.palustris and L. subrotundus, in their naturalhabitats may grow up to between 1 m and1.5m in stem length (Fig. 2), have abundantfoliage and are considered as good foragespecies. It is also known that certainspecies, along with feeding value, have goodsoil-fixing value and could be used as weed-cleaning crops (4). Most of the collectedspecies are mezo-xerophytes, but L. palustrisgrows on the moist and wet soils and usuallywell endures long flooding. So, theintraspecific variability of characters of theexamined Lathyrus species allows a selectionimprovement, pre-breeding and developing anovel protein-rich forage resource.

References(1)Vishnyakova MA, Burlyaeva MO (2006) Potential of household value and perspectives of using of Russian Chicklings. Agric Biol 6:85-97(2) Burlyaeva MO, Solovyeva AE, NikishkinaMA, Rasulova MA, Zolotov SV (2012) Species of the genus Lathyrus L. from N.I. Vavilov Institute (VIR) collection - the source of initial material for high-protein forage varieties breeding. LegumGroat Crop 4:62-70(3) Cherepanov SK (2007) Vascular Plants of Russia and Adjacent States (the Former USSR). Cambridge University Press, Cambridge(4) Rambaugh MD (1990) Special purpose forage legumes In: Janick J, Simon JE (eds) Advances in New Crops. Timber Press, Portland, USA, 183-190

Figure 2. Some Lathyrus species in their natural habitats in the Far East of Russian Federation: L. japonicus (above, left), L. davidii (above, right), L. subrotundus (below, left), L. palustris(below, right)

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On some biomorphological peculiarities of seedlings of Vavilovia formosa (Stev.) Fed. (Fabaceae)by Janna A. AKOPIAN1, Andrey A. SINJUSHIN2, Ivan G. GABRIELYAN1 and Gayane G. SHABOYAN1

Abstract: Wild perennial pea, Vavilovia formosa isa rare representative of the alpine flora. and itsgrowing in ex situ conditions is very complicated.The assumption that the border betweencataphylls and true leaves of perennial Fabeaecoincides with the border between wintering andannual plant parts is confirmed by observationson V. formosa samples grown from seeds inlaboratoryconditionsand in naturalhabitats.Key words: biomorphology, ex situconservation,ontogeny,Vavilovia formosa

Wild perennial pea, Vavilovia formosa (Stev.)Fed., is a rare representative of the alpine upper-mountain flora with distribution confined tosmall areas of moving detritus and screes. That iswhy its growing in ex situ conditions is verycomplicated. Мoderate air temperature, evenlower ground temperature, continuous hydration(from melting glaciers), together with sufficientaeration of the substrate, dryness of the upperlayers of the scree and a low concentration ofmineral salts in the soil with pH 6.5-7.0 areimportant for the successful growing of this plant(1, 2). Nevertheless, attempts to grow and studyV. formosa in other conditionscontinue.

The aim of this work was to study the earlystages of V. formosa ontogeny on samples grownfrom seeds in laboratory conditions. The seedlingmorphology was also studied in natural habitats(Geghama ridge, Mt. Sevsar, 2014). Seeds werecollected in the territory of Armenia from twopopulations: from the slopes of Geghama ridge(Mt. Sevsar, 3200-3300 m asl., 2002) and Syunikridge (Mt.Ukhtasar,3300-3350m asl., 2012).

The V. formosa seeds from the slopes of Mt.Sev Sar (Fig. 1a) are mostly elongated orroundish-oval, flattened (5.0 mm - 4.8 mm × 2.8mm - 3 mm × 2.4 mm -2.5 mm), while the seedsfrom the vicinity of Mt. Ukhtasar (Fig. 1b) arealmost spherical, less flattened (3.8 mm × 3.7mm - 3.8 mm × 2.8 mm - 2.9 mm).

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1National Academy of Sciences, Institute of Botany, Yerevan, Armenia ([email protected])2M.V. Lomonosov Moscow State University, Moscow, Russia

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Figure 1. Vavilovia seeds from two Armenian populations: Geghama ridge, Mt. Sevsar (a), and Syunik ridge, Mt. Ukhtasar (b)

Figure 2. A twelve-day old seedling of V. formosa with basal scale leaves (1-4)

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Seed surface is smooth, the color is dark olivewith black dots or spots. Immature seeds arebright brown. Seed hilum is broadly-ovate (0.7mm - 0.8 mm × 0.5 mm) with thin white coatingand a white line on the border of the cotyledons.Chalaza trail is dark brown orblack, inconspicuous.

Seeds were germinated in laboratory conditionsin Petri dishes on humid filter paper. After 5days, the swollen seeds were scarified, necessaryfor their germination 10-12 days after sowing.The seedlings were transplanted into smallcontainers with soil and gravel, with holes on thebottom and on the entire surface to ensureproper aeration of the root system. They werewateredsparingly,mainly from the pallet.

The root system of V. formosa seedlings usuallyhas a taproot and numerous lateral roots.However, in the early stages of germination, insome seedlings, the taproot ceases to grow andwhitish adventitious roots form near thecotyledonary node (Fig. 2). In otherplants, reducing the taproot and lateral rootformation to the fourth order was observed (Fig.3). The seedling hypocotyl had a length of 6 (7)mm, epicotyl 4 (5) mm, 2nd internode length 4(6) mm, 3rd 17 mm and 4th 20 (23) mm.

Four cataphylls (scaly leaves) are observed atthe base of the seedling (Fig. 2). The firstсataphyll is on the outer side of the epicotylsbend, 1.8 mm - 1.9 mm × 1.0 (1.2) mm, acutelylanceolate, entire, with a prominent mainvein, with membranous edges narrowly at thebase and widely at the top. The next twocataphylls are alternate, tridentate, with acutelylanceolate middle tooth, 1.9 mm × 1.8 mm (thesecond), 1.8 mm × 1.2 mm (the third). Thefourth cataphyll is greenish, 3.0 mm x 1.0 (0.9)mm, lateral teeth are 0.8 mm. It should bementioned that earlier three сataphylls werereported for the Vavilovia seedling (4). Leaflets offirst true leaves are obovate, about 5mm × 4mm, sparsely pubescent with white, softtrichomes from abaxial side and by edges andalmost glabrous from adaxial side. Stem and leafpetioles are covered with sparse, minute (0.03mm - 0.05 mm), narrowly clavate, reddishglandularhairs.

Seed germination of V. formosa is hypogeal.Naturally epicotyl, three next internodes and 3(4)cataphylls remain under moving scree layer. Theylack chlorophyll (Fig. 4), unlike seedlingsobtained in laboratory conditions (Fig. 2, 5a). Inall cataphyll axils, buds are formed, from whichin laboratory conditions the shoots of thefollowingordersare developed(Fig. 5A and 5B).

The assumption that the border betweencataphylls and true leaves of perennial Fabeaecoincides with the border between wintering andannual plant parts (3), is confirmed byobservations on V. formosa. In autumn in naturalhabitats, under a layer of scree, winteringhypogeal shoots of renewal develop from thebuds in axils of cataphylls (Fig. 5C). Annual aerialpart of the plant during the first year ofdevelopment represents unbranched orthotropicshoot up to 5-6 cm in height. By the end of thegrowing season, up to 10-13 leaves withelongated internodes are formed on a plant in thelaboratory, the same number of leaves and shortinternodes are observed under naturalconditions.

AcknowledgementsThe authors are grateful to Dr. M.E. Oganesianand Dr. A.G. Ghukasyan(Institute of Botany of National Academy of Sciences of Armenia) for kindly providing the seeds of Vavilovia formosa.

H

References(1) Fedorov AA (1939) Wild high mountain peas of Caucasus. Trud Biol Inst Arm S S R 1:39-79(2) Makasheva RK, Drozd AM, AdamovaOP, Golubev AA (1973) Perennial pea. Bull Appl BotGenet Plant Breed 51:44-56(3) Sinjushin AA (2013) Vavilovia, pea and other representatives of tribe Fabeae (Fabaceae): relation and similarity. Bull Appl Bot Genet Plant Breed 173:68-75(4) Sinjushin AA, Akopian JA (2011) On seedling structure in Pisum L., Lathyrus L. and Vavilovia Fed. (Fabeae: Fabaceae). Wulfenia 18:81-93

Figure 3. Root system of V. formosa in the first year of development under laboratory conditions: reduced main taproot (a1) and lateral roots (a2, a3 and a4)

Figure 4. A V. formosa plant in the first year of development on the moving scree slope of Mt. Sevsar, at the beginning of September

Figure 5. The V. formosa development features in 18 (A) and 33 days (B) after germinating in laboratory conditions and the basal part of the V. formosa plant in the first year of development in natural conditions at the beginning of September (C): seed (a) and hypogeal shoots of renewal (b)

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In vitro biotechnology approaches now available for ‘beautiful’ vavilovia (Vavilovia formosa)by Sergio J. OCHATT* and Catherine CONREUX

Abstract: Efficient in vitro propagation ofVavilovia formosa, plant regeneration fromcallus, protoplast isolation and culture todifferentiated callus of V. formosa were developedand its relative nuclear DNA content by flowcytometry was established. The summation ofbiotechnology tools now available should fosterevolutionary studies on the tribe Fabeae and, intime, V. formosa could become a potential sourceof novel agronomicadaptive traits.Key words: callus culture, evolutionarystudies, in vitropropagation,Vavilovia formosa

Vavilovia (Vavilovia formosa (Stev.) Fed.)material is as sparse as reports on it and acollaborative work started recently in severalEuropean laboratories (2, 3). Among othertopics, development of biotechnology tools forthis perennial herbaceous, endangered andprotected plant was undertaken. A few matureseeds were collected in the Caucasus, its mainarea of distribution, and in vitro cultures wereinitiated at Agritec Plant Research Ltd inŠumperk, Czech Republic, and the Institute ofBotany in Yerevan, Armenia. We now developedefficient in vitro propagation, plant regenerationfrom callus, protoplast isolation and culture todifferentiated callus of V. formosa, and we alsoestablished its relative nuclear DNA content byflow cytometry (1, 5).

Rapidly propagating, high quality shoots of V.formosa were produced after testing a range ofmedia with varying hormonal concentrations (6).The main problem was that shoots werestunted, enfeebled and incapable of rooting. Thiswas resolved by applyinga sequence of recurrentpassages on medium with low NAA and highBAP first, then to a similar BAP content withincreased NAA where multiplication rate wasreduced but shoot quality was increased.Thereafter, shoots were kept by cycling 4successive passages on medium with a balancedNAA/BAP content followed by 4 passages onmedium with the stronger cytokinin TDZ as solehormone.Shoots with with elongated internodes

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INRA, UMR1347 Agroécologie, Dijon , France ([email protected])

were thus consistently produced and 35% - 40%of them could be rooted (Fig. 1a) on mediacontaining full or half-strength Murashige andSkoog’s (4) formula with or without auxin (IBAor NAA).

Callus cultures were obtained from theinternodes and leaves of propagated shoots onmedium with 0.5 mg l-1 BAP and 1.0 mg l-1NAA, and shoots were also regenerated de novo(Fig. 1b), then rooted and transferred ex vitro.Finally, experiments were undertaken onprotoplast culture and regeneration from theleaves and stems of propagated shoots and alsofrom the rapidly proliferating calluses above. Todate, callus could be proliferated from theisolated protoplasts of all three tissue sources butonly callus-derived protoplasts were capable ofdifferentiation but, to date, de novo regeneration ofprotoplast-derivedplantshas remainedelusive.

Regenerated and propagated plants were true-to-type in terms of phenotype and relativenuclear 2C DNA content, which we determinedto be of 4.74 pg DNA.

The summation of biotechnology tools nowavailable should foster evolutionary studies onthe tribe Fabeae and, in time, V. formosa couldbecome a potential source of novel agronomicadaptive traits.

References(1) Doležel J, Bartoš J, Voglmayr H, Greilhuber J (2003) Nuclear DNA content and genome size of trout and human. Cytom A 51:127-128(2) Mikić AM, Smýkal P, Kenicer GJ, VishnyakovaMA, Sarukhanyan NG, Akopian JA, VanyanAG, Gabrielyan IG, Smýkalová I, Sherbakova E, ZorićL, Atlagić J, Zeremski-ŠkorićT, Ćupina BT, KrstićĐB, Jajić I, Antanasović S, ĐorđevićVB, MihailovićVM, Ivanov AL, Ochatt S, Ambrose MJ (2013) The bicentenary of the research on ‘beautiful’ vavilovia(Vavilovia formosa), a legume crop wild relative with taxonomic and agronomic potential. Bot J LinneanSoc 172:524-531(3) Mikić A, Smýkal P, Kenicer G, VishnyakovaM, Sarukhanyan N, Akopian JA, Vanyan A, GabrielyanI, Smýkalová I, Sherbakova E, ZorićL, AtlagićJ, Zeremski-SkorićT, Ćupina B, KrstićÐ, JajićI, Antanasović S, ÐorđevićV, MihailovićV, IvanovA, Ochatt S, Toker C, ZlatkovićB, Ambrose M (2014) Beauty will save the world, but will the world save beauty? The case of the highly endangered Vaviloviaformosa (Stev.) Fed. Planta, 240:1139-1146(4) Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. PhysiolPlant 15:473-497(5) Ochatt SJ (2008) Flow cytometry in plant breeding. Cytom A 73: 581-598(6) Ochatt S, Mousset-DéclasC, Rancillac M (2000) Fertile pea plants regenerate from protoplasts when calluses have not undergone endoreduplication. Plant Sci 156:177-183

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Figure 1. Some in vitro responses obtained with Vavilovia formosa: shoots with elongated internodes, the middle one shows rooting (a), and shoot regeneration from internode culture (b)

a b

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Nutritive value of grass pea (Lathyrus sativus)by Tihomir ČUPIĆ1*, Marijana TUCAK1 and Aleksandar MIKIĆ2

Abstract: Grain legumes are a rich source ofhigh quality food for human and animalconsumption. Their cultivation has beenconstrained by water deficit, high or lowacidity of soil and flowering sensitivity totemperature. Landraces of grass pea have alarge genetic potential for importantproduction traits including tolerance todrought and flood, winter-hardiness andtolerance to diseases and insects. They are arich source of protein, carbohydrates as wellas isoflavones. Our future research on grasspea will focus on further increasing yield andcreating lines without the ODAP toxinwhich could be of increasing importance asthe climate changes.Key words: grass pea, landraces, Lathyrussativus, nutritive value, ODAP

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1Agricultural Institute Osijek, Osijek, Croatia ([email protected])2Institute of Field and Vegetable Crops, Novi Sad, Serbia

The genus Lathyrus has 187 species andsubspecies (1). Of these, only a small numberare used in food and feed. Only grass pea(Lathyrus sativus L.) is cultivated and used ashuman food, forage for livestock or as asuitable crop for spreading green manure inextreme environmental conditions. Acrossthe world it has different names, but themost common is grass pea. In different partsof the world people use different names orsynonyms like (5) chickling pea (UK andNorth America), almora (Spain), khesari orbatura (India), alverjas (Venezuela), gilban(Sudan), guaya (Ethiopia), matri(Pakistan), gessette (France), pisello bretonne(Italy), sikirica or tijarica (Croatia), grah poljak(Bosnia and Herzegovina) and sastrica(Serbia). Botanists have identified a closeconnection between L. sativus (grass pea) andL. cicer (dwarf chickling) and a possibility ofcommon origin. The archaeobotanicalevidence has shown that L. sativus and L. cicerhave been grown on the Iberian Peninsuladuring the Neolithic (6) from where theyspread throughout the Mediterranean. Theevidence shows the L. sativus is probably theoldest cultivated crop in Europe (3).

The interest for production of grass peahas varied during human history and wasinfluenced by wars or other natural disasterslike drought, floods, high temperatures, etc.The last great exploitation of grass pea inEurope has happened during the SecondWorld War. Inhabitants in dry Mediterraneanareas sowed undemanding grass pealandraces in order to feed themselves andkeep from hunger. Because of this, manyresearchers have begun to explore this highlyadaptable legume. Its small demands on theground, combined with tolerance to droughtand other stress conditions (stagnantwater, tolerance to high soil acidity andalkalinity) makes it a plant species that willbecome more and more important as theclimate changes.

The main ingredients in grass pea grainsare carbohydrates with a share of about 55%and about 28% of protein of grain drymatter (DM). The rest of the material aremineral components (4%) and oil (about2%). The differences in the composition ofgrain are influenced by weatherconditions, soil composition and the types oflandraces that are used in specific geographicregions. The biological value of grass peaprotein is estimated at 70% of the biologicalvalue of soy protein, while the digestibility isapproximately equal. The most commonprotein in the structure of the grass pea seedis globulin (50% - 66%). Albumin values varywidely depending on the environmentalconditions (14% - 26%), while glutenin(about 15 %) and prolamin (5% - 6%) havethe lowest percentage. Of all the aminoacids, grass pea is the richest in lysine (4.5% -9.6%) and threonine (2.6% -5.1 %), while, like other legumes, it is poorwith cysteine (about 1%) and methionine(about 0.8%). The predominantcarbohydrate in grass pea is starch, with33.0% - 43.9%, followed by fiber whichcomprises about 7%. Total sugars varydepending on location and year (2.7% -5.2%), which is reflected in the water-solubleoligosaccharides (1.9% - 3.3%). The mostcommon oligosaccharid in the seed isstachyose, accounting for 1.1 g 100 g-1 to1.7 g 100 g-1, while the shares of verbascoseand raffinose are lower with an average of0.77 g 100 g-1 and 0.39 g 100 g-1, respectively.Oligosaccharides belong to the prebioticgroup and as such promote the growth andproliferation of bifidobacteria in the colonand have a beneficial effect on health and theprevention of cancer.

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Figure 1. One of the grass pea local landraces from Dalmatia: in bloom (above) and seeds (below)

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Grasspea, like most species of the genusLathyrus, contains anti-nutritionalcomponents (4) such as trypsininhibitors, lectins, tannins, phytic acid andthe water soluble non-protein amino acidODAP (oxalyl di-amino propanoic acid).Prolonged and excessive intake of ODAP(neurotoxin) causes lathyrism that can causeuncomfortable muscle spasms and certainneurological problems because of theinhibition of lysyl oxidase that directly affectsthe spinal cord. Lathyrism seems to occurwhen the grass pea ratio in food is at least25% continously over 1.5 to 6 months. Thetraditional varieties of grass pea contain 0.5%- 2.5% of β-ODAP. Our present findingssuggest that European genotypes havesignificantly less neurotoxins as compared toAfrican and Asian. However, the level ofneurotoxins is not only a genetic trait but isalso influenced by environmental factors.These have a significant effect on the level ofa ODAP. It has been established that thestress caused by drought increases the levelof neurotoxins and soil salinity reduces it.However, there is evidence in the landracesof grass pea that people, selecting by color offlower and seeds, unconsciously chose plantswith less ODAP and tannins and createdlocal populations in Dalmatia andHerzegovina, which have been used as foodfor many years. Laboratory tests are currentlyconducted in order to find genotypes with alow concentration of neurotoxins anddetermine agronomic factors that will reducethe level of ODAP below 0.10%.Similarly, studies are conducted on otheranti-nutritional compounds to obtain linesthat would be suitable for the creation ofnew more cultivated genotypes suitable forconsumption. Thermal treatment (hightemperature) can also inactivate most anti-nutritional compounds which enables thegrass pea to be used for humanconsumption.

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Legume Perspectives Issue 1 • January 2013

Apart from the above mentioned anti-nutritional substances, grass pea is abundantin phytoestrogens that appear in more than300 plant species of which legumesconstitute a major part. The total phenolcontent represents the potential value ofantioxidant activity of each population.Isoflavones are part of the isoflavonoidesgroup that has a characteristic chemicalstructure on which the physiological activityof individual isoflavones depends. Theantioxidant activity of isoflavones highlightspossible potential for lowering cholesterollevels, prevention of prostate cancer andbreast cancer, osteoporosis, cardiovasculardisease. It also relieves the symptoms ofmenopause. Two of the most important andmost researched sources of isoflavones aresoybean (from grain) and red clover (fromthe whole plant), but other legumes such asgrass pea can also be a good source ofisoflavones. Grass pea is a legume that isconsumed the most in Ethiopia, India andBangladesh. The amount of isoflavones inthe grain varies depending on thegenotype, geographic area of growth andproduction conditions. In the trialsconducted with grass pea accessions fromDalmatia the share of isoflavones wasaround 20 mg 100 g-1 DM. It was found thatsome populations with multi-colored seedhad significantly more total isofalvones, evenover 100 mg 100 g-1 DM. In mostpopulations an absence of genistein wasdetermined. The presence of genistein wasfound in one population, and is a valuablesource of potential inhibitory activity ofprotein-tyrosine kinases, which can reducethe possibility of cancer cell growth.Formononetin has the largest isoflavoneshare in the grass pea. The conductedanalysis has shown the presence formonetinin all populations (9.5 mg 100 g-1 DM onaverage). One local population contained aten times higher proportion of daidzein andis very promising material for furtherstudy, if the results replicate and confirm itas a stable quantitative trait. Bioactivecompounds in most legumes vary dependingon the genotype, the weather and otherenvironmental conditions. This opens newopportunities for further research related toindividual isoflavone content in grass pea.

Apart from good nutritional values, thegrasspea should potentially achieve high andstable grain yield in order to expandproduction. The current yield potential inlarge scale production ranges from 900kg ha-1 to 1500 kg ha-1, while yields in theUnited States in the production of inoculantsachieve quantities of 2000 kg ha-1. In the areaof Bosnia and Herzegovina, from 2008 to2009, experiments that were performedproduced the highest average yield of 3422kg ha-1 of grass pea grain with intensive useof agronomy (2). The results of this studyshow that the genetic potential for the yieldof grass pea is great and that usingappropriate agricultural technology needs tobe maximized. Identification of high-yieldand low concentrated deliverance grasspealines, should be increase the production inthe future.■

References:(1) Allkin R, Goyder DJ, Bisby FA, White RJ (1983) List of Species and Subspecies. The Vicieae, Vicieae Database Project 1, 4-11(2) Gatarić Đ, Radić V, Đurić B, Kovačević Z, Lakić Ž (2009) Morphological and biological characteristics of grass pea (Lathyrus sativus L.) seed, eco-type from Petrovo Polje. Agro-knowl J10:2:31-38(3) Kislev ME (1989) Origin of the cultivation of Lathyrus sativus and L. cicera (Fabaceae). Econ Bot43:262-270(4) Mikić A, Perić V, Đorđević V, Srebrić M, Mihailović V (2009) Anti-nutritional factors in some grain legumes. Biotechnol Anim Husb 25:1181-1188(5) Mikić A, Mihailović V, Ćupina B, Đurić B, Krstić Đ, VasićM, Vasiljević S, Karagić Đ, Đorđević V ( 2011) Towards the re-introduction of grass pea (Lathyrus sativus) in the West Balkan Countries: The case of Serbia and Srpska (Bosnia and Herzegovina). Food Chem Toxicol 49:650-654(6) Peña-Chocarro L, Peña LZ (1999) History and traditional cultivation of Lathyrus sativus L. and Lathyrus cicera L. in the Iberian peninsula. Veg Hist Archaeobot 8:49-52


The effect of cooking and enzymatic digestion on phenolic content of grass pea seeds flour extractsby Nemanja STANISAVLJEVIĆ1*, Živko JOVANOVIĆ1, Tihomir ČUPIĆ2, Jovanka LUKIĆ3, Jovanka MILJUŠ Jovanka MILJUŠ ĐUKIĆ1, Svetlana RADOVIĆ4 and Aleksandar MIKIĆ5

Abstract: Legume seeds are a rich source ofa wide variety of bioactive non-nutrientcompounds, including phenolicantioxidants, dominantly located in the seedcoat. Total phenolic content of methanolicand water extracts of non-processed, cookedand in vitro enzymatically digested seed flourof five grass pea (Lathyrus sativus) landraceswas studied. There was no significantdifference in phenolics content in non-processed grass pea seed flour. However, ourresults shown that cooking and enzymaticdigestion strongly enhanced the release ofphenolic compounds in methanolic extractsof all analyzed cultivars.Key words: bioactive compounds, cookingquality, grass pea, landraces, phenols

IntroductionLegume seeds are a rich source of minerals

and many nutrient compounds, includingsugars, starch, certain fatty acids andproteins. These seeds also contain a widevariety of bioactive non-nutrientcompounds, including phenolicantioxidants, dominantly located in the seedcoat.

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1University of Belgrade, Institute of Molecular Genetics and Genetic Engineering, Plant Molecular Biology Lab, Belgrade, Serbia ([email protected])2Agricultural Institute Osijek, Osijek, Croatia3University of Belgrade, Institute of Molecular Genetics and Genetic Engineering, Laboratory for Molecular Genetics of Industrial Microorganisms, Belgrade, Serbia4University of Belgrade, Faculty of Biology, Belgrade, Serbia 5Institute of Field and Vegetable Crops, Novi Sad, Serbia

Phenolic substances have an importantrole in the defense system of theseeds, which are exposed to oxidativedamage by many environmental factors, suchas light, oxygen, free radicals, and metal ions(6). These compounds are widely distributedin the plant kingdom and have been reportedto possess a considerable range of biologicaleffects-antioxidant, anti-inflammatory andvasodilatory action. It is known that plantphenolics might have the role in theprevention of coronary disease, cancer andage-related degenerative brain disorders.

Legume seeds are usually cooked in orderto increase palatability and improve edibility.All this process may bring about a numberof changes in physical and chemicalcharacteristics. There are many contradictorydata about the effect of cooking andenzymatic digestion on phenolic content(1, 5). There is also data that extractingsolvent significantly affects phenolic contentand antioxidant activity.

The aim of this study was to determine theextent to which conventional cookingmethods combined with gastrointestinal-simulated extraction of phenols, differ fromthe extraction of unprocessed grass pea seedflour with pure methanol.

Materials and methodsLegume samples. The grass pea (Lathyrus

sativus L.) seed samples were taken fromseveral local landraces from southernCroatia: L1-Dalmatinska zagora “PakovoSelo”, L2- Dalmatinska zagora “Drniš”, L3-Ravni kotari “Škrabinja“, L4-Dalmatinskazagora “Stankovci” and L5-Cetinska krajina“Tijarica”. Voucher specimens of thepopulation studied are deposited in theHerbarium of the Department of ForageCrops, Agricultural Institute Osijek, Croatia.

Chemicals and reagents. Folin-Ciocalteureagent (FC), gallic acid (GA), sodiumcarbonate, pancreatin and trypsin wereobtained from Sigma Chemical. Co. (St.Louis, MO, USA). All other chemicals wereof analytical grade.

Preparation of methanolic extracts from dry seeds.Dry grass pea seeds were ground to a finepowder in a basic mill to pass through a 60-mesh sieve. Pure methanol (2 ml) was addedin aliquot of the powder (0.2 g) and tubeswere shaken at 300 rpm at 4 ºC in the darkfor 24h. The extracts were centrifuged at5000 g for 5 min and the supernatants werecollected.

Preparation of methanolic extracts from cookedseed flour. Seed flour (0.2 g) was mixed with 2ml of water in tube, shaken and the sampleswere boiled for 45 min. The cooking waterwas evaporated under vacuum at 60 ºC andthe samples were freeze-dried. The methanolextraction was previously described.

Preparation of methanolic extracts from cookedand in vitro enzymatically digested seed flour. The invitro enzymatic digestion was performedaccording (2), with some modifications. Seedflour samples (0.2g) were cooked asprevoisuly described and mixed with salinesolution (pH = 2.0) containingNaCl, KCl, NaHCO3 and pepsin. Themixture digested at 37 ºC for 90 min. Thedistilled water containing 2% bile was addedand the mixture adjusted to pH = 8 withNaOH. After incubation for 10 min at 37 ºCin anaerobic condition, pancreatin was addedto final concentration of 1%, followed byincubation for 12 min at 37 ºC in anaerobiccondition. The extracts were thencentrifuged at 5000 g for 5 min, the liquidwas evaporated at 60 ºC under vacuum, andthe precipitate was freeze-dried. One groupof precipitate was resuspended in puremethanol and extracted as previouslydescribed. Another group was resuspendedin water and extracted to obtain aqueousextracts.

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Phenolic content/FC reducing capacity assay.Total phenol content was determined by aFolin-Ciocalteu (FC) assay (3), using gallicacid (GA) as a standard. Total phenoliccontent was expressed as GA equivalents(mg of GA g-1 samples).

Statistics. All the experiments were carriedout in triplicate and data were expressed asmeans ± standard deviations. The data weresubjected to ANOVA and Duncan’smultiple range tests using SPSS 19 softwareto determine significant differences at p <0.995.

Results and discussionAlthough the FC assay was not supposed

to characterize antioxidant activity, it seemsto be the one of the best for a roughdetermination of antioxidant activity of foodsamples. It was non-specific for phenols, asthe reagent can be reduced by many non-phenolic compounds. This assay was recentlyproposed for the measurement of totalreducing capacity. The phenolic content inmethanolic extracts of grass pea seeds flourranged from 0.25 mg GA g-1 for L1 to 0.37mg GA g-1 for L3 (Table 1), but with nosignificant difference. In general, thelandraces with colored seed coat have higherphenolic contents (6). In our case, the seedcoat color and phenolic content werecorrelated - the most pigmented L3 landraceexhibited the highest phenolic conent. Thereis a report on a negative correlation betweenseed size and total phenolic content in grasspea (4), but not in our case (r2 = -0.14).

Regarding the fact that legumes are usuallycooked for human nutrition, there is aninterest in determining the influence ofcooking on phenolic content. although thereare many contradictory data. Someexperiments showed that boiling significantlyalters phenolics content, but the changesdepended on the type of legume andprocessing conditions. The results obtainedfor our cooked extracts showed a consistentincrease in phenolic content after cooking.compared to the methanolic extracts (Table1). The reason for such increase isincomplete extraction of phenoliccompounds by the single extraction step thatwe applied.

From these results we can conclude thatcooking enhance the liberation of phenols.Methanolic extracts of cooked and in vitrodigested seed flour contained in somelandraces three times more phenols thanmethanolic extracts. Statistically significantdifferences (p < 0.05) were observed in allinvestigated landraces. The increase inphenolic content in cooked and in vitrodigested extracts over only cooked extractscould be attributed not only to liberation ofphenols, but also to liberation of smallpeptides soluble in methanol. These peptidescan interact with FC reagent, resulting innon-specific absorption at 765 nm. Similarresults were obtained on soybean, wheregastrointestinal extracts showed highervalues for total phenols than methanolicextracts.

ConclusionThis study showed that processing legume

seed flour by cooking and enzymaticdigestion strongly enhanced the release ofphenolic compounds. The effects ofprocessing were consistent in majority ofinvestigated grass pea landraces. Methanolicextraction of cooked and digested seedsyielded the highest phenolic content.

AcknowledgmentsThis work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Grant No. 173005 and ERA-NET-168 SEELEGUMES

References(1) Adefegha SA, Oboh G (2011) Cooking enhances the antioxidant properties of some tropical green leafy vegetables. Afr J Biotechnol10:632-639(2) Chen CY, Blumberg JB (2008) In vitro activity of almond skin polyphenols for scavenging free radicals and indicing quinine reductase. J Agric Food Chem 56:4427-4434(3) Julknene-Titto R (1985) Phenolic constituents in the leaves of northern willows: methods for the analysis of certain phenolics. Agric Food Chem33:213-217(4) Pastor-Cavada E, Juan R, Pastor JE, AlaizM, Viouque J (2009) Antioxidant activity of seed polyphenols in fifteen wild Lathyrus species from South Spain. LWT-Food Sci Technol 42:705-709(5)Starzynska-Janiszewska A, Stodolak B, JamrozM (2008) Antioxidant proprieties of extracts from fermented and cooked seeds of Polish cultivars of Lathyrus sativus. Food Chem 109:285-2926) Troszynska A, Ciska E (2002) Phenoliccompounds of seed coats of white and colouredvarieties of pea (Pisum sativum L.) and their total antioxidant activity. Czech J Food Sci 20:15-22

Table 1. Phenolic content/FC reducing capacity of seed flour extracts from grass pea landraces (mg of gallic acid g-1 sample); values are means of three replications (N = 3 ± SD); values marked by the same small case letter are not significantly different at p < 0.05 (Duncan test)

Landrace Methanolic extracts Cooked Cooked and digested

L1 0.27 ± 0.03a 0.72 ± 0.04b 0.89 ± 0.01c

L2 0.30 ± 0.02a 0.79 ± 0.02b 0.84 ± 0.04b

L3 0.37 ± 0.02a 0.91 ± 0.06b 1.04 ± 0.13b

L4 0.33 ± 0.01a 0.69 ± 0.02b 0.86 ± 0.05c

L5 0.25 ± 0.01a 0.58 ± 0.01b 0.76 ± 0.05c

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Screening for dietary phenolics and antioxidant capacity of faba bean (Vicia faba) and vetches (Vicia spp.)by Đorđe MALENČIĆ1*, Biljana KIPROVSKI1, Branko ĆUPINA1 and Aleksandar MIKIĆ2

Abstract: The phenolics in faba bean andvetches may act as antioxidants, therebyreducing the risk of atherosclerosis andcoronary heart disease, which can be causedby oxidation of low-density lipoproteins. Theresults of our study showed that theexamined Vicia species differed greatly intheir phenolics levels, as well as that thephenolics content should be considered asan important feature of Vicia species sincesome of its nutritive and pharmacologicaleffects.Key words: bioactive compounds, fababean, phenolics, vetches

Faba bean (Vicia faba L.) and vetches (Viciaspp.) belong to the family Fabaceae andrepresent indigenous species and traditionalfood in some parts of the Balkans. Althoughprimarily grown for their protein and vitamincontent and diuretic and lithontripticproperties, they also may be considered apotential biological source of dietaryphenolics. The phenolics in faba bean andvetches may act as antioxidants, therebyreducing the risk of atherosclerosis andcoronary heart disease, which can be causedby oxidation of low-density lipoproteins(2, 3). For the reasons of alimentary andpharmaceutical purposes, the aim of thisstudy was to select the Vicia populationswith higher phenolics content and increasedantioxidant activity.

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1University of Novi Sad, Faculty of Agriculture, Novi Sad, Serbia ([email protected])2Institute for Field and Vegetable Crops, Novi Sad, Serbia

The 70% aqueous acetone extracts of tenVicia species, grown as autochthonouspopulations in different regions ofSerbia, were used for determination of totalpolyphenols, tannins and proanthocyanidinsamounts (1). Amounts of total flavonoidswere estimated from MeOH : H2O :CH3COOH extracts (140 : 50 : 10) (4).Antioxidant activity was evaluated byDPPH-radical scavenging activity assay (5).Correlation between phenolic classescontents and antioxidant activity wasestablished by regression analysis.

The results showed that the examinedVicia species differed greatly in theirphenolics levels. The content of totalpolyphenols ranged from 160.2 mg catechin100 g-1 to 608.7 mg catechin 100 g-1 , whiletannins varied between 26.2 mg catechin100 g-1 and 297.9 mg catechin 100 g-1 of drymatter (DM). Flavonoids levels were muchlower, up to 0.15 mg rutine 100 g-1

DM, while the content of proanthocyanidinsranged from 5.5 mg leucoanthocyanidin100 g-1 DM to 92.4 mg leucoanthocyanidin100 g-1 DM. The DPPH values varied widelybetween 21.1% and 89.6% of neutralizedradicals, which mainly correlated with totalpolyphenols and tannins contents, with rranging from 0.74 to 1.00 (Fig. 1 and Fig. 2).

The results obtained suggest that thephenolics content should be considered asan important feature of Vicia species, assome of its nutritive and pharmacologicaleffects could be attributed to theirpresence.

References(1) Hagerman A, Harvey-Mueller I, Makkar HPS (2000) Quantification of Tannins in Tree Foliage – A Laboratory Manual. FAO/IAEA, Vienna(2) Malenčić Đ, Popović M, Miladinović J (2007) Phenolic content and antioxidant properties of soybean (Glycine max (L.) Merr.) seeds. Mol 12:576-581 (3) Malenčić Đ, Cvejić J, Miladinović J (2012) Polyphenols content and antioxidant properties of colored soybean seeds from central Europe. J Med Food 15:89-95(4) Marckam KR (1989) Methods in Plant Biochemistry. Academic Press, London(5) Washida K, Abe N, Sugiyama Y, Hirota A (2007) Novel DPPH radical scavengers, demethylbisorbibutenolide and trichopyrone, from a fungus. Biosci Biotechnol Biochem71:1052-1057

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Figure 1. Correlation between DPPH and total polyphenols in Vicia species

Figure 2. Correlation between DPPH and total tannins in Vicia species

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SEELEGUMES: One genuine piece in the jigsaw puzzle of the European legume biodiversityby Branko ĆUPINA1* and Aleksandar MIKIĆ2

The project ERA-168 SustainablePreservation of Indigenous South East EuropeanLegumes and Their Traditional Food and FeedProducts (SEELEGUMES) was carried outwithin the SEE-ERA.NET Plus programmeand under the auspices of the SeventhFramework Programme (FP7) of theEuropean Union during 2011 and 2012.

Coordinated by Dr. Branko Ćupina fromthe Faculty of Agriculture of the Universityof Novi Sad, Serbia, SEELEGUMESgathered together 15 full and 4 associatepartners from 13 countries, namelyAlbania, Armenia, Bulgaria, Croatia, Finland,France, FYR of Macedonia, Germany,Greece, Romania, Serbia, Slovenia andSrpska (Bosnia and Herzegovina).

The wild and agricultural floras in all WestBalkan countries and other South EastEuropean (SEE) regions are rather rich inlegume species. Many of these have becameand nearly forgotten, remained a part of wildflora despite its great agronomic potential.The strategic goal of this project was toestablish an efficient, functional andsustainable network on the genetic resourcesof annual legume species in the SEEcountries and enable it to be fit into thesimilar existing EU and other internationalnetworks.

The SEELEGUMES project consisted oftwo workpackages (WP):

WP1 Gathering SEE legumes, focusing onexpeditions, mapping, collecting, ex situ andin situ preservation and establishing apassport database of the collected accessionsof diverse status, such as crop wildrelatives, weedy species and local landraces;

WP2 Describing SEE legumes, emphasizing aconventional and molecular characterisationof the collected species and an analysis oftheir nutritional and other values.

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1University of Novi Sad, Faculty of Agriculture, Novi Sad, Serbia ([email protected])2Institute for Field and Vegetable Crops, Novi Sad, Serbia

Along from numerous deliverables, such astwo workshops (Fig. 1 and Fig. 2) andnumerous scientific and popularpublications, the project inducedcollaborating with eminent individuals frommany other institutions, whose precious

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Figure 2. Participants of the Second SEELEGUMES Workshop, Thessaloniki, Greece, 23-26 September 2012

Figure 1. Participants of the First SEELEGUMES Workshop, Banja Luka, Republic of Srpska, Bosnia and Herzegovina, 19-21 October 2011

contributions are also presented in this issue.To rearrange a Beatles’ song: Oh, we’ve got bywith a great help from our friends!

Please visit the SEELEGUMES projectweb site at http://polj.uns.ac.rs/~seelegumes/index.html.

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Second International Legume Society Conference (ILS2) 2016: Legumes for a Sustainable WorldTróia, Portugal, 12-14 October 2016

The International Legume Society and the Instituto de Tecnologia Química e Biológica of theUniversidade Nova de Lisboa cordially invite you to join us at the Second International LegumeSociety Conference, scheduled from 12-14 October, 2016 at Tróia resort, in the vicinity ofLisbon, Portugal.

In a world urgently requiring more sustainable agriculture, food security and healthier diets the demandfor legume crops is on the rise. This growth is fostered by the increasing need for plant protein and forsound agricultural practices that are more adaptable and environmentally sensitive. Food, feed, fiberand even fuel are all products that come from legumes – plants that grow with low nitrogen inputs andin harsh environmental conditions. The Second Legume Society Conference will be held during 2016 -the United Nations’ International Year of Pulses. The goals of this UN International Year include:the encouragement of connections throughout the food chain that would better utilize pulse basedproteins; increase global production of pulses; better utilization of crop rotations; and to addresschallenges in the trade of pulses.

The conference will address the following themes: Legume Quality and Nutrition; FarmingSystems/Agronomy; Abiotic and Biotic Stress Responses and Breeding; Legume Genetic Resources;and New “Omics” Resources for Legumes. The health and environment benefits, as well as, themarketing of legumes will be transversal topics throughout the conference. Special attention willbe given to foster the interaction of researchers and research programs with different stakeholdersincluding farmers and farmer associations, seed/feed and food industries, and consumers. Forthis, the conference will also be the site of the Final Meeting of the EU-FP7 ABSTRESS project, theAnnual Meeting of EU-FP7 LEGATO project; and final dissemination events of EU-FP7-ERANetsMEDILEG and REFORMA. The results and conclusions from these four important research programswill be shared with conference attendees.

Please join us in beautiful Tróia, Portugal from 12-14 October, 2016! Plan now to include the SecondILS Conference in your busy agenda. Kindly share this information with any colleagues dealing withlegumes.

Diego Rubiales, on behalf of the Scientific Committee Pedro Fevereiro, Carlota Vaz Patto and Susana Araújo, on behalf of the Organizing Committee


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Local Organizers The Instituto de Tecnologia Química e Biológica / Universidade Nova de Lisboa (ITQB/UNL) will be responsible for the organization of the Conference, in cooperation with the International Legume Society. The official language of the Conference will be the English.

Conveners Pedro Fevereiro – [email protected] Carlota Vaz Patto - [email protected] Susana Araújo - [email protected]

Scientific Coordinator Diego Rubiales

Local Organizer Committee Ana Barradas – FertipradoCarlota Vaz Patto - ITQB/UNL Eduardo Rosa – Universidade de Trás-os-Montes e Alto Douro Isabel Duarte – Instituto Nacional de Investigação Agrária e Veterinaria (INIAV) Manuela Costa – Universidade do Minho Manuela Veloso – INIAV Marta Vasconcellos – Escola Superior de Biotecnologia, Universidade CatólicaNuno Almeida - ITQB/UNL Pedro Fevereiro –ITQB/UNL Sofia Duque – ITQB/UNL Susana Araújo - ITQB/UNL Susana Leitão - ITQB/UNL

Scientific Committee Adrian Charlton: FERA, York, UK Aleksandar Mikić: Institute of Field and Vegetable Crops, Novi Sad, Serbia Charles Brummer: University of California, Davis, USA Diego Rubiales: Institute for Sustainable Agriculture, CSIC, Spain Fred Stoddard: University of Helsinki, Finland Gerard Duc: INRA-Dijon, France Judith Lichtenzveig: Curtin University, Australia Kevin McPhee: USDA-ARS, Washington State University, USA Michael Abberton: IITA, Nigeria Noel Ellis: ICRISAT, India Paolo Annichiarico: CRA-FLC, Lodi, Italy Pedro Fevereiro: ITQB/UNL, Portugal Stephen E. Beebe: CIAT, Colombia Shiv Kumar Agrawal: ICARDA, Syria Tom Warkentin: University of Saskatchewan, Canada

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Venue

The conference will be held at Troia Design Hotel (http://www.troiadesignhotel.com) in Tróia in the vicinity of Lisbon, Portugal. Tróia is a beautiful sand peninsula dividing the Sado River from the Atlantic Ocean.

The nearest airport is the Lisbon International Airport, about 50 Km away. Shuttles will be made available from and to Lisbon International Airport.

During the period of Roman occupation, date from the 1st century to the 6th century AD, Tróia was an island of Sado delta, called Ácala Island.

Sado Estuary Nature Reserve, where dolphins swim, and the Serra da Arrábida Natural Park, where a full developed Mediterranean forest can be seen, are two of the main natural attractions nearby Tróiapeninsula.

The Tróia Golf Championship Course is considered the best course in Portugal in the categories of difficulty and variety. It also stands in 20th place in the list of the best golf courses in Europe drawn up by the Golf World magazine.

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Tentative Programme

October 11th, 2016Morning-Afternoon: Satellite projects meetings Evening: Conference Registration

October 12th, 2016 08:00 Registration; 09:00 Welcome addresses; 09:45 Session 1 (Opening plenary)11:15 Coffee break11:45 Sessions 2 & 312:45 Lunch14:30 Sessions 2 & 316:30 - 19:00 Sessions 4 & 5 20:45 Third International Legume Football Cup

October 13th, 2016 9:00 Session 611:15 Coffee break11:45 Sessions 7 & 812:45 Lunch14:30 Sessions 7 & 816:00 Coffee break 16:30 International Legume Society Assembly 20:45 Third International Legume Football Cup

October 14th, 2016 09:00 Session 911:15 Coffee break11:45 Sessions 10 & 1112:45 Lunch14:30 Sessions 10 & 1116:00 Coffee break16:30 Session 12 (Closing plenary) 20:00 Farewell Dinner

October 15th, 2016 Satellite projects meetings

Bem-vindo ao Tróia, amigos dos leguminosas!

EVENTS

18th Symposium of the European Grassland FederationGrassland and Forages in High Output Dairy Farming Systems

Wageningen, the Netherlands, 15-18 June 2015http://www.europeangrassland.org/events.html

Symposium of the Protein Crops Working Group, Protein Crops Section, and Spanish Association for Legumes

Plant Proteins for the FuturePontevedra, Spain, 4-7 May 2015

http://www.symposiumproteincrops.org/

Symposium of the Fodder Crops and Amenity Grasses SectionBreeding in a World of Scarcity

Ghent, Belgium, 14-17 September 2015http://www.eucarpia-fcag2015.be/

IN MEMORIAM

Professor Reid G. PalmerDr. Reid Palmer, born on June 21, 1941, in Pemberville, Ohio, a

USDA Research Geneticist at the Iowa State University and affiliateProfessor of Agronomy after retiring in 2012, passed away inAmes, Iowa, on August 17, 2014.

Reid has been giving his precious support to the idea of globallegume science networking, conceived within the Grain LegumeTechnology Transfer Platform (GL-TTP), in which he activelyparticipated as one of the most remarkable representatives of theinternational soybean research community.

The International Legume Society and the Legume Perspectivesjournal editors and readers most sincerely commiserate with Reid’swidow Nuray and his numerous cousins, friends and colleagues.

May our dear Reid tend his beloved soybean forever and in peaceon the fields of heaven.

Professor Reid G. Palmer giving his presentation on hybrid soybean at the Second GL-TTP Workshop, Novi Sad, Serbia, November 28, 2008

UN International Year of Pulses - 2016http://www.iyop.net/en/

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SSupport Legume Society and become its sponsor [email protected]

LEGUME SOCIETY INSTITUTE OF FIELD AND VEGETABLE CROPS, NOVI SAD, SERBIAwww.ils.nsseme.com www.nsseme.com

CONSIGLIO PER LA RICERCA E LA SPERIMENTAZIONE INSTITUT NATIONAREGIONAL PROGRAMME FOR COOPERATION WITH SOUTHEAST EUROPE

http://www.see-era.net/

UNIVERSITY OF NOVI SAD SPANISH MINISTRY OF SCIENCE AND INNOVATIONFACULTY OF AGRICULTURE, NOVI SAD, SERBIA SPANISH NATIONAL RESEARCH COUNCIL

http://polj.uns.ac.rs/ www.csic.es

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